5-aminolevulinic acid derivatives, methods for their preparation and uses thereof

ABSTRACT

There are provided conjugates comprising 5-aminolevulinic acid (5-ALA), an aldehyde and a carboxylic acid (e.g., a histone deacetylase inhibitor), compositions comprising same, methods for their preparation and uses thereof for treatment of cancer and anemia, and for inducing erythropoiesis.

FIELD OF THE INVENTION

The present invention relates to drug conjugates comprising5-aminolevulinic acid (5-ALA), an aldehyde and a carboxylic acid (e.g.,histone deacetylase inhibitor (HDACI)), compositions comprising them anduses thereof for treatment of cancer and anemia and for inducingerythropoiesis.

BACKGROUND OF THE INVENTION

Photodynamic therapy (PDT) is a promising type of non-invasive therapy,and favorable results of PDT have been reported in glioma patients [1].In general, PDT involves at least the components of a photosensitizerand irradiating light (at a wavelength appropriate for thephotosensitizer). The light causes the photosensitizer to damage andkill cells and tissues exposed to the irradiated light. Aminolevulinicacid (ALA)-PDT therapy is based on the administration of aminolevulinicacid (ALA), the natural precursor for protoporphyrin IX (PpIX)biosynthesis, which is a potent natural photosensitizer. Irradiation ofALA treated cancer cells in the presence of oxygen results in generationof singlet oxygen that is toxic to the tumor [2]. ALA-PDT is the mostused phototherapy application for skin cancers such as basal cellcarcinomas and in addition, ALA based photo-diagnosis is used forintraoperative dissection of gliomas and bladder tumors [3]. A maindisadvantage of ALA-PDT is the hydrophilic nature of ALA, which limitsits ability to penetrate deeper into tissue layers. One solution toimprove the poor penetrability of ALA is to increase its lipophilicityusing ALA esters such as methyl-ALA and hexyl-ALA [4].

It has been shown that ALA acyloxyalkyl ester prodrugs are hydrolyzedinto ALA which induces PpIX synthesis [5]. The advantage of theseALA-prodrugs stems from their ability to induce cancer cells death byboth PDT and non-PDT mechanisms at doses lower than ALA.

Pharmacological inhibition of histone deacetylase (HDAC) activity bysmall organic molecules (HDACIs) could provide therapeutic benefit to avariety of diseases and disorders [6]. Histone deacetylase inhibitoryprodrugs which are small molecular weight fatty acids, that uponintracellular hydrolytic degradation release acids and aldehydes havebeen described [7-9]. These compounds have been shown to modulate geneexpression, induce histone hyperacetylation, differentiation, andapoptosis of cancer cells in vitro, ex-vivo and in vivo [10-12].

Anemia is a common, and sometimes the major, clinical symptom of a widevariety of pathological conditions [13]. Its severity is determined bytwo parameters: the number of red blood cells (RBC) and their hemoglobin(Hb) content. Erythropoietin (EPO) is the major stimulating hormone ofred blood cell formation (erythropoiesis). It is produced, principally,in the kidney, and its levels are controlled by tissue oxygen tension.In bone marrow, it promotes the survival, proliferation and maturationof the erythroid progenitors and precursor cells thus leading to theelevation of red cell mass and Hb level. Several types of anemia can beameliorated by EPO. It has been used primarily in cases of anemia due toEPO-insufficiency associated with chronic renal failure, and also foranemia due to different cases. EPO has also been introduced as apreemptive/prophylactic treatment for patients undergoing electivesurgery, to avoid heterologous blood transfusion. Although the latterprocedure is safe in most cases, it is not without risk due toblood-borne pathogens, immunomodulating factors and severe allergicreactions. In an alternative approach, autologous blood is used fortransfusions; patients are phlebotomized prior to surgery, RBC harvestedand stored, and then used during or following the operation.Nevertheless, treatment with EPO, the costs of which are high, increasesthe yield of the RBC harvested and ameliorates the transient phase ofmild anemia that might follow. Moreover, in placebo-controlled studies,administration of EPO prior to surgery increased Hb level at and aftersurgery and significantly lowered the need for blood transfusions [14].Although meta analyses on the effect of EPO on cancer patients indicatedincrease in life quality benefit, it also showed increased risks forthromboembolic and cardiovascular events.

Thus, there is a need in the art for improved methods for treatment ofanemia, that are less toxic and less expensive. In particular, it wouldbe advantageous to have drugs that can replace EPO or reduce the dose ofEPO required for treating anemia.

SUMMARY OF THE INVENTION

The present invention provides drug conjugates comprising5-aminolevulinic acid (ALA), an aldehyde and a carboxylic acid that mayfunction as a histone deacetylase inhibitor (HDACI). These conjugatesmay serve as co-drugs which release a plurality of active species invivo. The novel drug conjugates may be used, for the treatment orprevention of cancer in PDT-dependent and/or PDT-independent (non-PDT)treatments, as well as for cosmetic uses.

In addition the present invention provides novel uses for both the noveland known compounds. According to some embodiments, the presentinvention provides drug conjugates (co-drugs) comprising (i) ALA, (ii)an aldehyde and (iii) a carboxylic acid that may function as a histonedeacetylase inhibitor (HDACI) for the treatment of anemia and/or for theinduction of erythropoiesis.

According to some embodiments, there are provided compounds representedby the structure of formula (I):

wherein

-   -   R¹ is        -   (a) a C₁-C₂₀ straight, branched, saturated or unsaturated or            cyclic alkyl, wherein said alkyl may be unsubstituted or            substituted with a phenyl, halogen, or oxygen;        -   (b) —CH₂CH₂—CO—CH₂—NH—R³; or        -   (c) —CH(NHCOCH₃)CH₂—SH;    -   R² is H or a C₁-C₂₀ straight, branched, saturated or        unsaturated, or cyclic alkyl, wherein said alkyl may be        unsubstituted or substituted with a phenyl, halogen, or oxygen;        and    -   R³ is H or a nitrogen protecting group;    -   or a pharmaceutically acceptable salt thereof;    -   with the proviso that when R¹COO is derived from pivalic,        butyric or valproic acid, R² is not H or CH₃;

including salts, polymorphs, optical isomers, geometrical isomers,enantiomers, diastereomers, and mixtures thereof.

The group R¹C(═O)—O— is derived from a carboxylic acid of formulaR¹C(═O)OH, wherein R¹ is as defined above. In some embodiments,R¹C(═O)O— is derived from a carboxylic acid selected from the groupconsisting of pivalic, butyric, valeric, hexanoic, 4-phenylbutyric,4-phenylacetic, heptanoic, octanoic, decanoic, and retinoic acid.Currently preferred carboxylic acids are butyric, octanoic, decanoic,valeric or retinoic acid, and particularly preferred are butyric acid oroctanoic acid. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, R¹ is a C₁-C₁₀ straight, branched,saturated or unsaturated or cyclic alkyl, wherein said alkyl may beunsubstituted or substituted with a phenyl, halogen, or oxygen. Infurther embodiments, R¹ may be a C₃-C₁₀ straight, branched, saturated orunsaturated or cyclic alkyl, wherein said alkyl may be unsubstituted orsubstituted with a phenyl, halogen, or oxygen. In further embodiments,R¹ may be a C₁₀-C₂₀ straight, branched, saturated or unsaturated orcyclic alkyl, wherein said alkyl may be unsubstituted or substitutedwith a phenyl, halogen, or oxygen. In some embodiments, R¹ is selectedfrom the group consisting of methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonanyl and decyl, with propyl and heptyl beingcurrently preferred.

The group R²—(CH)—O— is derived from an aldehyde of formula R²C(═O)H,wherein R² is as defined above. According to some embodiments, thealdehyde is formaldehyde (in which case R² is H). According to otherembodiments, the aldehyde is acetaldehyde (in which case R² is CH₃).According to other embodiments, the aldehyde is propionaldehyde (inwhich case R² is CH₂CH₃). According to other embodiments, the aldehydeis butyrladehyde (in which case R² is CH₂CH₂CH₃). In furtherembodiments, R² is a C4-C10 straight, branched, saturated or unsaturatedor cyclic alkyl, wherein said alkyl may be unsubstituted or substitutedwith a phenyl, halogen, or oxygen. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, R³ is H. In further embodiments, R³ is anitrogen protecting group selected from Boc and Cbz.

Non-limiting examples of compounds of formula (I) according to thepresent invention are compounds of formula (A), (B) and (C):

Compounds A-C are represented by the following chemical names:

-   1-(Octanoyloxy)ethyl-5-amino-4-oxopentanoate (A);-   1-(Butyryloxy)butyl-5-amino-4-oxopentanoate (B);-   1-(Butyryloxy)propionyl-5-amino-4-oxopentanoate hydrochloride (C).

In some embodiments, the compounds of formula (A) to (C) are provided inthe form of pharmaceutically acceptable salts, preferably thehydrochloride (HCl) salts.

In some embodiments, the compound of formula (I) is in the form of anacid addition salt. The salt may be derived from a pharmaceuticallyacceptable acid selected from the group consisting of hydrochloric,hydrobromic, sulfuric, methane sulfonic, benzene sulfonic, naphthylsulfonic, acetic, tartaric, maleic and malic acids. Currently preferredacid addition salts are hydrochloric acid (HCl) salts.

According to additional embodiments, there is provided a pharmaceuticalcomposition comprising a compound of formula (I), and a pharmaceuticallyacceptable carrier or excipient. In some embodiments, the pharmaceuticalcomposition is in a form suitable for oral administration, intravenousadministration by injection, topical administration, dermatologicaladministration, administration by inhalation, or administration via asuppository.

In additional embodiments, there is provided a pharmaceuticalcomposition comprising a compound of formula (I) that is in a formsuitable for topical or dermatological administration, and thecomposition further comprises a topically or dermatologically acceptablecarrier or excipient.

Each compound of the present invention may be formulated in such apharmaceutical composition, with each possibility representing aseparate embodiment of the present invention. In a currently preferredembodiment, the pharmaceutical composition of the present inventioncomprises a compound of formula (I) or (I-a) wherein R¹COO is derivedfrom retinoic acid, and further comprises a topically ordermatologically acceptable carrier or excipient.

According to some embodiments, the compounds of formula (I) areco-drugs, that may be hydrolyzed in-vivo, to produce one or more activecompounds that may exert a biological effect. In some embodiments, thecompounds of formula (I) may be hydrolyzed to 5-ALA, a carboxylic acidand an aldehyde. In some embodiments, the carboxylic acid may be aninhibitor of histone deacetylase (that is, the carboxylic acid may be anHDACI).

According to some embodiments, the present invention relies at least inpart on the finding that derivatives of 5-ALA and HDACI (which may bederived from co-drug compounds represented by Formula (I) upon in-vivohydrolysis), improve the neoplastic activities of dependent photodynamictherapy (PDT) and independent photodynamic therapy (non-PDT) andspecifically affect cancer cells with a substantially lower effect onnormal cells.

According to some embodiments, the acyloxymethyl ester co-drug(s) ofFormula (I) are highly active in PDT-independent (non-PDT) anti cancertreatment. In some embodiments, the acyloxyalkyl co-drugs of formula (I)elicit high photodynamic-dependent antineoplastic activity. In someembodiments, co-drugs represented by compounds of formula (I) arehydrolyzed in-vivo to yield 5-ALA and a carboxylic acid, wherein thecarboxylic acid may be an HDACI. In some embodiments, the HDACI is anoctanoic acid.

According to some embodiments, there is thus provided a method for thetreatment or prevention of cancer, comprising the step of administeringto a subject in need thereof the compound of formula (I) orpharmaceutical composition comprising the same.

According to some embodiments, the treatment or prevention of cancer isselected from photodynamic therapy (PDT), non-photodynamic therapy(non-PDT), or a combination thereof. In some embodiments, when R² is H,the cancer treatment comprises non-photodynamic therapy (non-PDT). Insome embodiments, when R² is a C₁-C₂₀ straight, branched, saturated orunsaturated, or cyclic alkyl, and the cancer treatment comprisesphotodynamic therapy (PDT).

According to further embodiments, there is provided a method for thetreatment or prevention of cancer, comprising the step of administeringto a subject in need thereof a therapeutically effective amount ofoctanoic acid or a therapeutically acceptable salt thereof.

According to some embodiments, there are provided compounds of formula(I-a), for use in the treatment or prevention of anemia, wherein thecompound(s) of formula (I-a) are represented by the following structure:

wherein

-   -   R¹ is        -   (a) a C₁-C₂₀ straight, branched, saturated or unsaturated or            cyclic alkyl, wherein said alkyl may be unsubstituted or            substituted with a phenyl, halogen, or oxygen;        -   (b) —CH₂CH₂—CO—CH₂—NH—R³; or        -   (c) —CH(NHCOCH₃)CH₂—SH;    -   R² is a C₁-C₂₀ straight, branched, saturated or unsaturated, or        cyclic alkyl, wherein said alkyl may be unsubstituted or        substituted with a phenyl, halogen, or oxygen; and    -   R³ is H or a nitrogen protecting group;    -   or a pharmaceutically acceptable salt thereof;    -   including salts, polymorphs, optical isomers, geometrical        isomers, enantiomers, diastereomers, and mixtures thereof.

The group R¹C(═O)—O— is derived from a carboxylic acid of formulaR¹C(═O)OH, wherein R¹ is as defined above. In some embodiments,R¹C(═O)O— is derived from a carboxylic acid selected from the groupconsisting of pivalic, butyric, valeric, hexanoic, 4-phenylbutyric4-phenylacetic, heptanoic, octanoic, decanoic, and retinoic acid.Currently preferred carboxylic acids are butyric, octanoic, decanoic,valeric or retinoic acid, and aparticularly preferred are butyric acidor octanoic acid. Each possibility represents a separate embodiment ofthe present invention.

According to some embodiments, R¹ is a C₁-C₁₀ straight, branched,saturated or unsaturated or cyclic alkyl, wherein said alkyl may beunsubstituted or substituted with a phenyl, halogen, or oxygen. Infurther embodiments, R¹ may be a C₅-C₁₀ straight, branched, saturated orunsaturated or cyclic alkyl, wherein said alkyl may be unsubstituted orsubstituted with a phenyl, halogen, or oxygen. In further embodiments,R¹ may be a C₁₀-C₂₀ straight, branched, saturated or unsaturated orcyclic alkyl, wherein said alkyl may be unsubstituted or substitutedwith a phenyl, halogen, or oxygen. In some embodiments, R¹ is selectedfrom the group consisting of methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonanyl and decyl.

The group R²—(CH)—O— is derived from an aldehyde of formula R²C(═O)H,wherein R² is as defined above. According to some embodiments, thealdehyde is formaldehyde (in which case R² is H). According to otherembodiments, the aldehyde is acetaldehyde (in which case R² is CH₃).According to other embodiments, the aldehyde is propionaldehyde (inwhich case R² is CH₂CH₃). According to other embodiments, the aldehydeis butyrladehyde (in which case R² is CH₂CH₂CH₃). In furtherembodiments, R² is a C₄-C₁₀ straight, branched, saturated or unsaturatedor cyclic alkyl, wherein said alkyl may be unsubstituted or substitutedwith a phenyl, halogen, or oxygen. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, R³ is H. In further embodiments, R³ is anitrogen protecting group selected from Boc and Cbz.

Non-limiting examples of compounds of formula (I-a) according to thepresent invention are compounds of formula (A), (B) and (C) as shownabove, and further compounds of formula (D) to (G), as definedhereinbelow:

Compounds D-G are represented by the following chemical names:

-   1-(butyryloxy)ethyl 5-amino-4-oxopentanoate (D);-   (pivaloyloxy)methyl 5-amino-4-oxopentanoate (E);-   (butyryloxy)methyl 5-amino-4-oxopentanoate (F);-   1-(pivaloyloxy)ethyl 5-amino-4-oxopentanoate (G).

In some embodiments, the compounds of formula (D) to (G) are provided inthe form of pharmaceutically acceptable salts, preferably thehydrochloride (HCl) salts.

In some embodiments, the compound of formula (I-a) is in the form of anacid addition salt. The salt may be derived from a pharmaceuticallyacceptable acid selected from the group consisting of hydrochloric,hydrobromic, sulfuric, methane sulfonic, benzene sulfonic, naphthylsulfonic, acetic, tartaric, maleic and malic acids. Currently preferredacid addition salts are hydrochloric acid (HCl) salts.

According to further embodiments, there is provided a method forinducing erythropoiesis, comprising the step of administering to asubject in need thereof a compound represented by the structure offormula (I-a), or a pharmaceutical composition comprising such compound.

According to some embodiments, there are provided compounds of formula(I-a), for use in the treatment or prevention of anemia and/or forinduction of erythropoiesis.

According to some embodiments, the compounds represented by formula(I-a) are co-drugs, that may be hydrolyzed in-vivo, to produce one ormore active compounds that may exert a biological effect. In someembodiments, the compounds of formula (I-a) may be hydrolyzed to 5-ALA,a carboxylic acid and an aldehyde. In some embodiments, the carboxylicacid may be an inhibitor of histone deacetylase (that is, the carboxylicacid may be an HDACI). In some embodiments, the HDACI is an octanoicacid. In some embodiments, the HDACI is Butyric acid.

According to further embodiments, the compounds represented by formula(I-a) may be used to induce differentiation of erythrocytes and toinduce erythropoiesis.

According to some embodiments, there are further provided pharmaceuticalcompositions comprising a therapeutically effective amount of at leastone compound represented by the structure of formula (I-a). Suchpharmaceutical compositions may be used, in some embodiments, for thetreatment or prevention of anemia in a subject. In some embodiments,such pharmaceutical compositions may be used for induction oferythropoiesis in a subject.

According to further embodiments, there is provided a method for thetreatment or prevention of anemia, comprising the step of administeringto a subject in need thereof a combination comprising an HDAC-inhibitorand 5-aminolevulinic acid (5-ALA) or an ester thereof, wherein the esteris a methyl or a hexyl ester.

In some embodiments, the pharmaceutical composition is in a formsuitable for oral administration, intravenous administration byinjection, topical administration, dermatological administration,administration by inhalation, or administration via a suppository.

In additional embodiments, there is provided a pharmaceuticalcomposition comprising a compound of formula (I-a) that is in a formsuitable for topical or dermatological administration, and thecomposition further comprises a topically or dermatologically acceptablecarrier or excipient.

In addition to the aspects and embodiments described above, furtheraspects and embodiments will become apparent by reference to the figuresand by study of the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments are illustrated in referenced figures. Dimensions ofcomponents and features shown in the figures are generally chosen forconvenience and clarity of presentation and are not necessarily shown toscale. The figures are listed below.

FIG. 1 shows in schematic form a process for the synthesis of compoundsI and (I-a), according to some embodiments;

FIGS. 2A-B show the effect of compounds of formula (I-a) on thesynthesis of protoporphirin IX (PpIX) in K562 cells. FIG. 2A—shows arepresentative histogram depicting flow cytometry analysis of PpIXexpression. FIG. 2B shows pictures of cells expressing PpIX;

FIGS. 3A-C show the effect of various tested compounds on the activityand synthesis of porphobilinogen deaminase (PBGD) in K562 cells. FIG. 3Ashows a bar graph of the relative activity of PGBD compared to control.FIG. 3B shows a western blot analysis of the expression of PBGD in thecells. FIG. 3C shows a bar graph of quantitative RT-PCT analysis of theexpression of PBGD mRNA in the cells, in response to various treatments.FIGS. 3D-E show the effect of various treatments on the synthesis ofFerrochelatase in K562 cells. FIG. 3D shows a western blot analysis ofthe expression of Ferrochelatase in the cells. FIG. 3E shows a FACSanalysis of the expression of Ferrochelatase in the cells;

FIGS. 4A-E show the effect of various tested compounds on the synthesis(amount) of hemoglobin. FIG. 4A shows a bar graph of the fold increasein total heme content. FIG. 4B shows a bar graph of the fold increase intotal heme content in response to treatment with various compounds offormula (I-a) (D, 1-(butyryloxy)ethyl 5-amino-4-oxopentanoate (AlaAcBu),compound D, above; C, 1-(Butyryloxy)propionyl-5-amino-4-oxopentanoatehydrochloride, compound C, above; B,1-(Butyryloxy)butyl-5-amino-4-oxopentanoate Hydrochloride, compound B,above). FIG. 4C shows a bar graph of the fold increase in mRNAexpression of α-globin under various treatments. FIG. 4D show a Westernblot analysis of the expression levels of α-globin. FIG. 4E show a bargraph of the expression levels of α-globin protein;

FIGS. 5A-B show the effect of various tested compounds ondifferentiation of erythroblasts. FIG. 5A shows a bar graph of therelative expression of glycophorin A. FIG. 5B shows a representativehistogram depicting flow cytometry analysis of glycophorin A expression;

FIGS. 6A-B show the effect of various compounds on K562 cellproliferation. FIG. 6A shows a bar graph illustrating the mitochondriaactivity (% of control) under various treatments as measured by MTTassay. FIG. 6B shows FACS analysis of the cells under differentexperimental conditions;

FIG. 7 shows TEM pictographs of cells under various experimentalconditions. Panels A-B, control; Panels C-D: cells treated with BA;Panels E-F: cells treated with AlaAcBu. Solid arrows: central stackingof mitochondria; dashed arrows: multiple vacuolar system precedingnuclear extrusion.

FIGS. 8A-B show results of in-vivo experiments testing the effect ofcompound of Formula (I-a) on Doxorubicin induced anemia in mice. FIG. 8Ais a scheme of the experiment protocol, showing the treatment regime ofthe tested compounds of the tested groups: Group I (treated withDoxorubicin alone) and Group II (treated with Doxorubicin and a compoundof Formula (I-a). FIG. 8B is a bar graph of the amount of hemoglobin(mg/mL) in blood retrieved from Balb-c mice treated with doxorubicin(DOX) or doxorubicin in combination with a compound of formula (I-a)((in this example, AlaAcBu), (marked in the figure as DOX+1-a)).

FIGS. 9A-B show line graphs demonstrating the effect of octanoic acid onthe activity of HDAC. FIG. 9A shows the effect of octanoic acid on theactivity of HDAC in glioblastoma cell line U251. FIG. 9B shows theeffect of compound of formula (I-a) on the activity of HDAC inglioblastoma cell line U251.

DETAILED DESCRIPTION OF THE INVENTION Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below. It is to be understood that theseterms and phrases are for the purpose of description and not oflimitation, such that the terminology or phraseology of the presentspecification is to be interpreted by the skilled artisan in light ofthe teachings and guidance presented herein, in combination with theknowledge of one of ordinary skill in the art.

An “alkyl” group refers to any saturated or unsaturated aliphatichydrocarbon, including straight-chain, branched-chain and cyclic alkylgroups. It is understood that an “unsaturated alkyl” refers to an“alkenyl” or “alkynyl” group, as defined herein, and that a “cyclicalkyl group” refers to a cycloalkyl group as defined herein. In oneembodiment, the alkyl group has 1-20 carbons designated here asC₁-C₂₀-alkyl. In another embodiment, the alkyl group has 1-10 carbonsdesignated here as C₁-C₁₀-alkyl. In another embodiment, the alkyl grouphas 1-5 carbons designated here as C₁-C₅-alkyl. The alkyl group may beunsubstituted or substituted by one or more groups selected fromhalogen, hydroxy, alkoxy, aryloxy, alkylaryloxy, heteroaryloxy, oxo,cycloalkyl, phenyl, heteroaryl, heterocyclyl, naphthyl, amino,alkylamino, arylamino, heteroarylamino, dialkylamino, diarylamino,alkylarylamino, alkylheteroarylamino, arylheteroarylamino, acyl,acyloxy, nitro, carboxy, carbamoyl, carboxamide, cyano, sulfonyl,sulfonylamino, sulfinyl, sulfinylamino, thiol, C₁ to C₁₀ alkylthioarylthio, or C₁ to C₁₀ alkylsulfonyl groups. Any substituent can beunsubstituted or further substituted with any one of theseaforementioned substituents.

An “alkenyl” group refers to an aliphatic hydrocarbon group containingat least one carbon-carbon double bond including straight-chain andbranched-chain alkenyl groups. In one embodiment, the alkenyl group has2-8 carbon atoms designated here as C₂-C₈-alkenyl. In anotherembodiment, the alkenyl group has 2-6 carbon atoms in the chaindesignated here as C₂-C₆-alkenyl. Exemplary alkenyl groups includeethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl,heptenyl, octenyl, cyclohexyl-butenyl and decenyl. The alkenyl group canbe unsubstituted or substituted through available carbon atoms with oneor more groups defined hereinabove for alkyl.

An “alkynyl” group refers to an aliphatic hydrocarbon group containingat least one carbon-carbon triple bond including straight-chain andbranched-chain. In one embodiment, the alkynyl group has 2-8 carbonatoms in the chain designated here as C₂-C₈-alkynyl. In anotherembodiment, the alkynyl group has 2-6 carbon atoms in the chaindesignated here as C₂-C₆-alkynyl. Exemplary alkynyl groups includeethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl,heptynyl, octynyl and decynyl. The alkynyl group can be unsubstituted orsubstituted through available carbon atoms with one or more groupsdefined hereinabove for alkyl.

A “cycloalkyl” group refers to any saturated or unsaturated (e.g.,cycloalkenyl, cycloalkynyl) monocyclic or polycyclic group. In someembodiments, the cycloalkyl group has 3-20 carbon atoms designated hereas C₃-C₂₀-cycloalkyl. The cycloalkyl group may be monocyclic, fusedbicyclic or tricyclic, etc. Non-limiting examples of cycloalkyl groupsare cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.Non-limiting examples of cycloalkenyl groups include cyclopentenyl,cyclohexenyl and the like. The cycloalkyl group can be unsubstituted orsubstituted with any one or more of the substituents defined above foralkyl.

As used herein, the term “nitrogen protecting group” (P) refers to agroup which may be attached to a nitrogen atom to protect said nitrogenatom from participating in a reaction and which may be readily removedfollowing the reaction. The nitrogen protecting group can be an acidlabile protecting group, a base labile protecting group, or a protectinggroup that is removable under neutral conditions. Non-limiting examplesof nitrogen-protecting groups are silyl protecting groups [Si(R)₃wherein R is alkyl, aryl, aralkyl, and the like], acyl groups such asacetyl (COCH₃), benzoyl, 2-bromoacetyl, 4-bromobenzoyl,tert-butylacetyl, carboxaldehyde, 2-chloroacetyl, 4-chlorobenzoyl,α-chlorobutyryl, 4-nitrobenzoyl, o-nitrophenoxyacetyl, phthalyl,pivaloyl, propionyl, trichloroacetyl, and trifluoroacetyl; amide groupssuch as acetamide and the like; sulfonyl groups such as benzenesulfonyl,and p-toluenesulfonyl; carbamate groups of the formula —C(O)O—R whereinR is for example methyl, ethyl, t-butyl, benzyl, phenylethyl,CH₂═CH—CH₂, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl(Boc), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, and thelike. Other suitable nitrogen protecting group include, but are notlimited to: benzyl, formyl, phenylsulfonyl, (Fmoc),p-nitrobenzenesulfoethoxycarbonyl propargyloxycarbonyl, picolinyl,prenyl, o-nitrobenzyloxy methyl, 4-methyoxyphenoxymethyl,guaiacolmethyl, siloxymethyl, such as triisopropylsiloxymethyl,2-cyanoethyoxymethyl, 2-quinolinylmethyl, dichloroacetyl,trichloroacetyl and 2-[4-nitrophenyl]ethylsulfonate, as well as benzyl,p-methoxy benzyl and trityl. Each possibility represents a separateembodiment of the invention. A currently preferred protecting group isBoc. Another currently preferred protecting group is Cbz.

Other examples of nitrogen-protecting groups are described by C. B.Reese and E. Haslam, “Protective Groups in Organic Chemistry, “J. G. W.McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4,respectively, and T. W. Greene and P. G. M. Wuts, “Protective Groups inOrganic Synthesis,” 2nd ed., John Wiley and Sons, New York, N.Y., 1991,Chapters 2 and 3, each of which is incorporated herein by reference.

All references cited herein are hereby incorporated by reference intheir entirety, as if fully set forth herein.

As used herein, the term “salt” encompasses both basic and acid additionsalts, including but not limited to carboxylate salts or salts withamine nitrogens, and include salts formed with the organic and inorganicanions and cations discussed below. Further encompassed by the term aresalts formed by standard acid-base reactions with basic groups (such asamino groups) and organic or inorganic acids. Such acids includehydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric,acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic,pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric,lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic,naphthylsulfonic, sorbic, picric, benzoic, cinnamic, and the like.

All stereoisomers of the compounds of the present invention arecontemplated, either in admixture or in pure or substantially pure form.These compounds can have asymmetric centers at any of the atoms.Consequently, the compounds can exist in enantiomeric or diastereomericforms or in mixtures thereof. The present invention contemplates the useof any racemates (i.e. mixtures containing equal amounts of eachenantiomers), enantiomerically enriched mixtures (i.e., mixturesenriched for one enantiomer), pure enantiomers or diastereomers, or anymixtures thereof. The chiral centers can be designated as R or S or R,Sor d,D, l,L or d,l, D,L. In addition, several of the compounds of thepresent invention may contain one or more double bonds. The presentinvention intends to encompass all structural and geometrical isomersincluding cis, trans, E and Z isomers and optical isomers, independentlyat each occurrence. The thioamides of the present invention occur in twoisomeric forms known as atropisomers, due to hindered rotation aroundthe thioamide bond. These isomers can interconvert in solution andratios may vary at different conditions including temperature, pH,solvent, concentration, and the like.

The term “treating” as used herein refers to abrogating, inhibiting,slowing or reversing the progression of a disease or condition,ameliorating clinical symptoms of a disease or condition or preventingthe appearance of clinical symptoms of a disease or condition. The term“preventing” is defined herein as barring a subject from acquiring adisorder or disease or condition.

The term “treatment of cancer” is directed to include at least one ofthe following: a decrease in the rate of growth of the cancer (i.e. thecancer still grows but at a slower rate); cessation of growth of thecancerous growth, i.e., stasis of the tumor growth, and, in preferredcases, the tumor diminishes or is reduced in size. The term alsoincludes reduction in the number of metastases, reduction in the numberof new metastases formed, slowing of the progression of cancer from onestage to the other and a decrease in the angiogenesis induced by thecancer. In most preferred cases, the tumor is totally eliminated.Additionally included in this term is lengthening of the survival periodof the subject undergoing treatment, lengthening the time of diseasesprogression, tumor regression, and the like.

The term “therapeutically effective amount” refers to the amount of acompound being administered which provides a therapeutic effect for agiven condition and administration regimen, specifically an amount whichrelieves to some extent one or more of the symptoms of the disorderbeing treated.

As used herein, the term “introducing” refers to the transfer ofmolecules/compounds, into a target site, that may include, for example,a cell, a tissue, an organ, and the like. The molecules can be“introduced” into the target cell(s) by any means known to those ofskill in the art. Introduction into the cells may be passive (forexample, by incubating the cells with the compounds). Introduction intothe cells may take use of various agents that are able tomediate/facilitate/allow the entrance of the compound into the cell. Thecells may be selected from isolated cells, tissue cultured cells, celllines, cells present within an organism body, and the like.

As referred to herein, the term “HDAC” is directed to histonedeacetylase or lysine deacetylase. Histone deacetylase(s) or lysinedeacetylase are a class of enzymes that remove acetyl group(s) from ane-N-acetyl lysine amino acid on a histone or other proteins. Its actionis opposite to that of histone acetyltransferase.

As referred to herein, the term “HDACI” is directed to histonedeacetylase inhibitor(s). HDACI are compounds which are able to inhibitthe activity of histone deacetylase or lysine deacetylase.

As referred to herein, the terms “ALA” and “5-ALA” may interchangeablybe used and are directed to 5-aminolevulinic acid.

As referred to herein, the terms “co-drug(s)” and “drug conjugate(s)”may interchangeably be used. The terms are directed to a single moleculecompound, which upon in-vivo processing (for example, by hydrolysis) mayyield two or more separate compounds/reagents, each may be active invivo. The processing of the co-drug may be performed in vivo, forexample, within cell, within a tissue, within an organ, and the like.The co-drug may be inactive when administered, but may be converted invivo to two or more active compound(s)/components. In some embodiments,the co-drug may be formulated to a pharmaceutical composition.

As referred to herein, the term “mixture” is directed to a mixture oftwo or more separate compounds/reagents that may be present in the samecomposition and may be administered in combination (simultaneously orsequentially).

According to some embodiments, there are provided compounds representedby the structure of formula (I):

wherein

-   -   R¹ is        -   (a) a C₁-C₂₀ straight, branched, saturated or unsaturated or            cyclic alkyl, wherein said alkyl may be unsubstituted or            substituted with a phenyl, halogen, or oxygen;        -   (b) —CH₂CH₂—CO—CH₂—NH—R³; or        -   (c) —CH(NHCOCH₃)CH₂—SH;    -   R² is H or a C₁-C₂₀ straight, branched, saturated or        unsaturated, or cyclic alkyl, wherein said alkyl may be        unsubstituted or substituted with a phenyl, halogen, or oxygen;        and    -   R³ is H or a nitrogen protecting group;    -   or a pharmaceutically acceptable salt thereof;    -   with the proviso that when R¹COO is derived from pivalic,        butyric or valproic, R² is not H or CH₃;    -   including salts, polymorphs, optical isomers, geometrical        isomers, enantiomers, diastereomers, and mixtures thereof.

In some embodiments, the group R¹C(═O)—O— is derived from a carboxylicacid of formula R¹C(═O)OH, wherein R¹ is as defined above. In someembodiments, R¹C(═O)O— is derived from a carboxylic acid selected fromthe group consisting of pivalic, butyric, valeric, hexanoic,4-phenylbutyric, 4-phenylacetic, heptanoic, octanoic, decanoic, andretinoic acid. Currently preferred carboxylic acids are butyric,octanoic, decanoic, valeric or retinoic acid, and aparticularlypreferred are butyric acid or octanoic acid. Each possibility representsa separate embodiment of the present invention.

According to some embodiments, R¹ is a C₁-C₂₀ straight or branched chainalkyl, a C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl or a C₃-C₂₀ cycloalkyl,wherein said alkyl, alkenyl, alkynyl or cycloalkyl may be unsubstitutedor substituted with a phenyl, halogen, or oxygen.

According to some embodiments, R¹ is a C₁-C₁₀ straight, branched,saturated or unsaturated or cyclic alkyl, wherein said alkyl may beunsubstituted or substituted with a phenyl, halogen, or oxygen. Infurther embodiments, R¹ may be a C₃-C₁₀ straight, branched, saturated orunsaturated or cyclic alkyl, wherein said alkyl may be unsubstituted orsubstituted with a phenyl, halogen, or oxygen. In further embodiments,R¹ may be a C₁₀-C₂₀ straight, branched, saturated or unsaturated orcyclic alkyl, wherein said alkyl may be unsubstituted or substitutedwith a phenyl, halogen, or oxygen. In some embodiments, R¹ is selectedfrom the group consisting of methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonanyl and decyl, with propyl and heptyl beingcurrently preferred.

The group R²—(CH)—O—

in formula I, which bonded to the R¹C(═O)—O— group, is derived from analdehyde of formula R²C(═O)H, wherein R² is as defined above. Accordingto some embodiments, the aldehyde is formaldehyde (in which case R² isH). According to other embodiments, the aldehyde is acetaldehyde (inwhich case R² is CH₃). According to other embodiments, the aldehyde ispropionaldehyde (in which case R² is CH₂CH₃). According to otherembodiments, the aldehyde is butyrladehyde (in which case R² isCH₂CH₂CH₃).

In further embodiments, R² is a C₄-C₁₀ straight, branched, saturated orunsaturated or cyclic alkyl, wherein said alkyl may be unsubstituted orsubstituted with a phenyl, halogen, or oxygen. In further embodiments,R² is a C₁-C₂₀ straight or branched chain alkyl, a C₂-C₂₀ alkenyl, aC₂-C₂₀ alkynyl or a C₃-C₂₀ cycloalkyl, wherein said alkyl, alkenyl,alkynyl or cycloalkyl may be unsubstituted or substituted with a phenyl,halogen, or oxygen. Each possibility represents a separate embodiment ofthe present invention.

According to some embodiments, R³ is H. In further embodiments, R³ is anitrogen protecting group selected from Boc and Cbz.

Non-limiting examples of compounds of formula (I) according to thepresent invention are compounds of formula (A), (B) and (C):

Compounds A-C are represented by the following chemical names:

-   1-(Octanoyloxy)ethyl-5-Amino-4-oxopentanoate (A);-   1-(Butyryloxy)butyl-5-amino-4-oxopentanoate (B);-   1-(Butyryloxy)propionyl-5-amino-4-oxopentanoate hydrochloride (C).

In some embodiments, the compounds of formula (A) to (C) are provided inthe form of pharmaceutically acceptable salts, preferably thehydrochloride (HCl) salts.

According to some embodiments, the compound of formula (I) is a co-drug,wherein upon its hydrolysis (for example, within a target cell) ishydrolyzed to one or more separate compounds/reagents that may exert abiological effect in the target cell. For example, upon introduction ofa compound of formula (I) to a cell, the compound may be hydrolyzed (forexample by cellular esterases) to produce 5-ALA, a carboxylic acidcompound and an aldehyde compound. In some embodiments, one or more ofthe separate compounds may be active in the cell, that is, they mayexert a biological effect in the cell. According to further embodiments,the administration/introduction of the co-drug to the cell may result inenhanced and improved activity of the compounds as compared tointroduction of the separate compounds into the cells, when not on thesame molecule (co-drug).

According to some embodiments, the carboxylic acid may be a C₁-C₂₀straight, branched, saturated or unsaturated or cyclic alkyl, whereinthe alkyl may be unsubstituted or substituted with a phenyl, halogen oroxygen, -carboxylic acid. For example, the carboxylic acid may be aHOOC—CH₂CH₂—CO—CH₂—NH—R³, wherein R³ may be H or a nitrogen protectinggroup. For example, the carboxylic acid may be HOOC—CH(NHCOCH₃)CH₂—SH.In some embodiments the carboxylic acid may inhibit histone deacetylaseactivity (that is, the carboxylic acid may be an HDACI).

According to some embodiments, the aldehyde may be formaldehyde. Forexample, the aldehyde may be a C₁-C₂₀ straight, branched, saturated orunsaturated, or cyclic alkyl.

According to some embodiments, upon introduction of a compound ofFormula (I) into a cell, the co-drug may be hydrolyzed to produce a5-ALA, octanoic acid and formaldehyde. For example, upon introduction ofa compound of Formula (I) into a cell, the co-drug may be hydrolyzed toproduce a 5-ALA, octanoic acid and acetaldehyde. For example, uponintroduction of a compound of Formula (I) into a cell, the co-drug maybe hydrolyzed to produce a 5-ALA, decanoic acid and formaldehyde. Forexample, upon introduction of a compound of Formula (I) into a cell, theco-drug may be hydrolyzed to produce a 5-ALA, decanoic acid andacetaldehyde. For example, upon introduction of a compound of Formula(I) into a cell, the co-drug may be hydrolyzed to produce a 5-ALA,valeric acid and formaldehyde. For example, upon introduction of acompound of Formula (I) into a cell, the co-drug may be hydrolyzed toproduce a 5-ALA, valeric acid and acetaldehyde. For example, uponintroduction of a compound of Formula (I) into a cell, the co-drug maybe hydrolyzed to produce a 5-ALA, 4-phenylbutyric acid and formaldehyde.For example, upon introduction of a compound of Formula (I) into a cell,the co-drug may be hydrolyzed to produce a 5-ALA, 4-phenylbutyric acidand acetaldehyde. For example, upon introduction of a compound ofFormula (I) into a cell, the co-drug may be hydrolyzed to produce a5-ALA, hexanoic acid and acetaldehyde. For example, upon introduction ofa compound of Formula (I) into a cell, the co-drug may be hydrolyzed toproduce a 5-ALA, retinoic acid and formaldehyde. For example, uponintroduction of a compound of Formula (I) into a cell, the co-drug maybe hydrolyzed to produce a 5-ALA, retinoic acid and acetaldehyde. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, when R¹COO is derived from hexanoic, R²is not H. According to other embodiments, when R¹COO is derived from4-phenylbutyric acid, R² is not H. According to other embodiments, whenR¹COO is derived from pivalic, butyric, valproic, hexanoic, or4-phenylbutyric acid, R² is not H. According to other embodiments, whenR¹COO is derived pivalic, butyric or valproic acids, R² is not CH₃.

According to some embodiments, the compounds represented by formula (I)may be used to inhibit growth of target cells and/or to kill targetcells. The target cells may be any type of cell. In some embodiments,the target cells are cancer cells. In some embodiments, the target cellsare non-cancer cells.

According to some embodiments, co-drug compounds represented by formula(I), may induce apoptosis of cells, such as, for example, cancer cellsor non-cancer cells, into which the co-drug compound has beenintroduced.

In further embodiments, co-drug compounds represented by formula (I),may induce down regulation of proteasome expression and activity incells, which may further lead to accumulation of ubiquitinated proteinsand rapid cell death. For example, the cell may be a cancer cell. Forexample, the cell may be non-cancer cell.

According to some embodiments, the acyloxyalkyl ester co-drugs offormula (I) may provide enhanced antineoplastic effect on cancer cellsunder photo-irradiation conditions (i.e., PDT dependent) as compared tothe effect achieved by the acyloxymethyl ester co-drugs of formula (I).Without wishing to be bound to theory or mechanism, under the PDTconditions, the formaldehyde that may be released in-vivo (within thecancer cells) from the acyloxymethyl ester co-drugs of formula (I) mayspecifically interrupt with PpIX biosynthesis, highest levels of whichenhance the PDT.

According to additional embodiments, the acyloxymethyl ester co-drugs offormula (I) provide enhanced antineoplastic effect on cancer cells undernon-PDT conditions as compared to the acyloxyalkyl ester co-drugs offormula (I). Without wishing to be bound to theory or mechanism, underthe non-PDT conditions, the formaldehyde that may be released in-vivo(within the cancer cells) from the acyloxymethyl ester co-drugs offormula (I) is able to enhance the production of reactive oxygen species(ROS) in the cells and consequently kill the cells (for example, within4-96 hours).

According to some embodiments, the compounds of formula (I) may thus beused in the treatment of various types of cancer, in both photodynamictherapy (PDT) and non photodynamic therapy (non-PDT).

Cancer is a disorder in which a population of cells has become, invarying degrees, unresponsive to the control mechanisms that normallygovern proliferation and differentiation. Cancer refers to various typesof malignant neoplasms and tumors, including metastasis to differentsites. Non-limiting examples of cancers which can be treated by thecompounds represented by the structure of formula (I) are ovariancancer, prostate cancer, breast cancer, skin cancer, melanoma, coloncancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer,Ewing's sarcoma, lymphoma, leukemia, multiple myeloma, head and neckcancer, kidney cancer, bone cancer, liver cancer and thyroid cancer.Specific examples of cancers which the compounds of the presentinvention are effective at treating or preventing are: adenocarcinoma,adrenal gland tumor, ameloblastoma, anaplastic tumor, anaplasticcarcinoma of the thyroid cell, angiofibroma, angioma, angiosarcoma,apudoma, argentaffinoma, arrhenoblastoma, ascites tumor cell, ascitictumor, astroblastoma, astrocytoma, ataxia-telangiectasia, atrial myxoma,basal cell carcinoma, bone cancer, bone tumor, brainstem glioma, braintumor, breast cancer, Burkitt's lymphoma, carcinoma, cerebellarastrocytoma, cervical cancer, cherry angioma, cholangiocarcinoma, acholangioma, chondroblastoma, chondroma, chondrosarcoma, chorioblastoma,choriocarcinoma, colon cancer, common acute lymphoblastic leukemia,craniopharyngioma, cystocarcinoma, cystofibroma, cystoma, cytoma,cutaneous T-cell lymphoma, ductal carcinoma in situ, ductal papilloma,dysgerminoma, encephaloma, endometrial carcinoma, endothelioma,ependymoma, epithelioma, erythroleukaemia, Ewing's sarcoma, extra nodallymphoma, feline sarcoma, fibroadenoma, fibrosarcoma, follicular cancerof the thyroid, ganglioglioma, gastrinoma, glioblastoma multiforme,glioma, gonadoblastoma, haemangioblastoma, haemangioendothelioblastoma,haemangioendothelioma, haemangiopericytoma, haematolymphangioma,haemocytoblastoma, haemocytoma, hairy cell leukemia, hamartoma,hepatocarcinoma, hepatocellular carcinoma, hepatoma, histoma, Hodgkin'sdisease, hypernephroma, infiltrating cancer, infiltrating ductal cellcarcinoma, insulinoma, juvenile angiofibroma, Kaposi sarcoma, kidneytumor, large cell lymphoma, leukemia, chronic leukemia, acute leukemia,lipoma, liver cancer, liver metastases, Lucke carcinoma, lymphadenoma,lymphangioma, lymphocytic leukemia, lymphocytic lymphoma, lymphocytoma,lymphoedema, lymphoma, lung cancer, malignant mesothelioma, malignantteratoma, mastocytoma, medulloblastoma, melanoma, meningioma,mesothelioma, metastatic cancer, Morton's neuroma, multiple myeloma,myeloblastoma, myeloid leukemia, myelolipoma, myeloma, myoblastoma,myxoma, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma,neurofibroma, neurofibromatosis, neuroglioma, neuroma, non-Hodgkin'slymphoma, oligodendroglioma, optic glioma, osteochondroma, osteogenicsarcoma, osteosarcoma, ovarian cancer, Paget's disease of the nipple,pancoast tumor, pancreatic cancer, phaeochromocytoma, pheochromocytoma,plasmacytoma, primary brain tumor, progonoma, prolactinoma, renal cellcarcinoma, retinoblastoma, rhabdomyo sarcoma, rhabdosarcoma, solidtumor, sarcoma, secondary tumor, seminoma, skin cancer, small cellcarcinoma, squamous cell carcinoma, strawberry haemangioma, T-celllymphoma, teratoma, testicular cancer, thymoma, trophoblastic tumor,tumourigenic, vestibular schwannoma, Wilm's tumor, or a combinationthereof.

According to some embodiments, there is thus provided a method for thetreatment or prevention of cancer, comprising the step of administrationto a subject in need thereof a compound represented by formula (I).

In some embodiments, the treatment or prevention of cancer is selectedfrom photodynamic therapy (PDT), non-photodynamic therapy (non-PDT), ora combination thereof.

According to some embodiments, when R² of the compound of formula (I) isH, the cancer treatment comprises non-photodynamic therapy (non-PDT).

According to further embodiments, when R² of the compound of formula (I)is a C₁-C₂₀ straight, branched, saturated or unsaturated, or cyclicalkyl, the cancer treatment comprises photodynamic therapy (PDT).

According to some embodiments, the enhanced effect of co-drugsrepresented by formula (I) in treatment of cancer cells, may allow theuse of lower molar concentration and/or lower light dose (inPDT-dependent treatment) to achieve a desired effect on the cancer cell(i.e., killing the cancer cell, inhibiting ifs growth, and the like).

According to some embodiments, octanoic acid is an HDACI that may beused to affect the growth/survival of cancer cells.

In some embodiments, there is thus provided a method for the treatmentor prevention of cancer, comprising the step of administering to asubject in need thereof a therapeutically effective amount of octanoicacid or a therapeutically acceptable salt thereof.

According to some embodiments, there are provided compounds of formula(I-a), for use in the treatment or prevention of anemia, wherein thecompound(s) of formula (I-a) are represented by the following structure:

wherein

-   -   R¹ is        -   (a) a C₁-C₂₀ straight, branched, saturated or unsaturated or            cyclic alkyl, wherein said alkyl may be unsubstituted or            substituted with a phenyl, halogen, or oxygen;        -   (b) —CH₂CH₂—CO—CH₂—NH—R³; or        -   (c) —CH(NHCOCH₃)CH₂—SH;    -   R² is a C₁-C₂₀ straight, branched, saturated or unsaturated, or        cyclic alkyl, wherein said alkyl may be unsubstituted or        substituted with a phenyl, halogen, or oxygen; and    -   R³ is H or a nitrogen protecting group;    -   or a pharmaceutically acceptable salt thereof;    -   including salts, polymorphs, optical isomers, geometrical        isomers, enantiomers, diastereomers, and mixtures thereof.

In some embodiments, the group R¹C(═O)—O— is derived from a carboxylicacid of formula R¹C(═O)OH, wherein R¹ is as defined above. In someembodiments, R¹C(═O)O— is derived from a carboxylic acid selected fromthe group consisting of pivalic, butyric, valeric, hexanoic,4-phenylbutyric 4-phenylacetic, heptanoic, octanoic, decanoic, andretinoic acid. Currently preferred carboxylic acids are butyric,octanoic, decanoic, valeric or retinoic acid, and aparticularlypreferred are butyric acid or octanoic acid. Each possibility representsa separate embodiment of the present invention.

According to some embodiments, R¹ is a C₁-C₂₀ straight or branched chainalkyl, a C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl or a C₃-C₂₀ cycloalkyl,wherein said alkyl, alkenyl, alkynyl or cycloalkyl may be unsubstitutedor substituted with a phenyl, halogen, or oxygen.

According to some embodiments, R¹ is a C₁-C₁₀ straight, branched,saturated or unsaturated or cyclic alkyl, wherein said alkyl may beunsubstituted or substituted with a phenyl, halogen, or oxygen. Infurther embodiments, R¹ may be a C₅-C₁₀ straight, branched, saturated orunsaturated or cyclic alkyl, wherein said alkyl may be unsubstituted orsubstituted with a phenyl, halogen, or oxygen. In further embodiments,R¹ may be a C₁₀-C₂₀ straight, branched, saturated or unsaturated orcyclic alkyl, wherein said alkyl may be unsubstituted or substitutedwith a phenyl, halogen, or oxygen. In some embodiments, R¹ is selectedfrom the group consisting of methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonanyl and decyl.

The group R²—(CH)—O—

in formula I-a, which bonded to the R¹C(═O)—O— group, is derived from analdehyde of formula R²—C(═O)H, wherein R² is as defined above. Accordingto some embodiments, the aldehyde is formaldehyde (in which case R² isH). According to other embodiments, the aldehyde is acetaldehyde (inwhich case R² is CH₃). According to other embodiments, the aldehyde ispropionaldehyde (in which case R² is CH₂CH₃). According to otherembodiments, the aldehyde is butyrladehyde (in which case R² isCH₂CH₂CH₃).

According to some embodiments, R² is a C₁-C₂₀ straight or branched chainalkyl, a C₂-C₂₀ alkenyl, a C₂-C₂₀ alkynyl or a C₃-C₂₀ cycloalkyl,wherein said alkyl, alkenyl, alkynyl or cycloalkyl may be unsubstitutedor substituted with a phenyl, halogen, or oxygen. In furtherembodiments, R² is a C₄-C₁₀ straight, branched, saturated or unsaturatedor cyclic alkyl, wherein said alkyl may be unsubstituted or substitutedwith a phenyl, halogen, or oxygen. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, R³ is H. In further embodiments, R³ is anitrogen protecting group selected from Boc and Cbz.

Non-limiting examples of compounds of formula (I-a) according to thepresent invention are compounds of formula (A), (B) and (C) as shownabove, and further compounds of formula (D) to (G), as shownhereinbelow:

Compounds D-G are represented by the following chemical names:

-   1-(butyryloxy)ethyl 5-amino-4-oxopentanoate (D);-   (pivaloyloxy)methyl 5-amino-4-oxopentanoate (E);-   (butyryloxy)methyl 5-amino-4-oxopentanoate (F);-   1-(pivaloyloxy)ethyl 5-amino-4-oxopentanoate (G).

In some embodiments, the compounds of formula (D) to (G) are provided inthe form of pharmaceutically acceptable salts, preferably thehydrochloride (HCl) salts.

According to some embodiments, a compound represented by formula (I-a)is a co-drug, wherein upon its hydrolysis (for example, within a targetcell) it is hydrolyzed to one or more separate compounds/reagents thatmay exert a biological effect in the target cell. For example, uponintroduction of a compound of formula (I-a) to a cell, the compound maybe hydrolyzed (for example by cellular esterases) to produce 5-ALA,carboxylic acid compound and aldehyde compound. In some embodiments, oneor more of the separate compounds may be active in the cell, that is,they may exert a biological effect in the cell. According to furtherembodiments, the administration/introduction of the co-drug to the cellmay result in enhanced and improved activity of the compounds ascompared to introduction of the separate compounds or a mixture thereofinto the cells, when not on the same molecule (co-drug).

According to some embodiments, the carboxylic acid may be a C₁-C₂₀straight, branched, saturated or unsaturated or cyclic alkyl, whereinthe alkyl may be unsubstituted or substituted with a phenyl, halogen oroxygen, -carboxylic acid. For example, the carboxylic acid may be aHOOC—CH₂CH₂—CO—CH₂—NH—R³, wherein R³ may be H or a nitrogen protectinggroup. For example, the carboxylic acid may be HOOC—CH(NHCOCH₃)CH₂—SH.In some embodiments, the carboxylic acid may inhibit Histone deacetylaseactivity (that is, the carboxylic acid may be an HDACI).

According to some embodiments, the aldehyde may be a C₁-C₂₀ straight,branched, saturated or unsaturated, or cyclic alkyl. In someembodiments, the aldehyde is not formaldehyde.

According to some embodiments, upon introduction of a compound ofFormula (I-a) into a cell, the co-drug may be hydrolyzed to produce5-ALA, butyric acid and acetaldehyde. For example, upon introduction ofa compound of Formula (I-a) into a cell, the co-drug may be hydrolyzedto produce 5-ALA, valeric acid and acetaldehyde. For example, uponintroduction of a compound of Formula (I-a) into a cell, the co-drug maybe hydrolyzed to produce 5-ALA, valproic acid and acetaldehyde. Forexample, upon introduction of a compound of Formula (I-a) into a cell,the co-drug may be hydrolyzed to produce 5-ALA, hexanoic acid andacetaldehyde. For example, upon introduction of a compound of Formula(I-a) into a cell, the co-drug may be hydrolyzed to produce 5-ALA,octanoic acid and acetaldehyde. For example, upon introduction of acompound of Formula (I-a) into a cell, the co-drug may be hydrolyzed toproduce 5-ALA, decanoic acid and acetaldehyde. For example, uponintroduction of a compound of Formula (I-a) into a cell, the co-drug maybe hydrolyzed to produce 5-ALA, 4-phenylbutyric acid and acetaldehyde.For example, upon introduction of a compound of Formula (I-a) into acell, the co-drug may be hydrolyzed to produce 5-ALA, phenylacetic acidand acetaldehyde. For example, upon introduction of a compound ofFormula (I-a) into a cell, the co-drug may be hydrolyzed to produce a5-ALA, retinoic acid and formaldehyde. For example, upon introduction ofa compound of Formula (I-a) into a cell, the co-drug may be hydrolyzedto produce a 5-ALA, retinoic acid and acetaldehyde. Each possibilityrepresents a separate embodiment of the present invention.

According to some embodiments, the compounds represented by the formula(I-a) are able to augment erythropoiesis. Without wishing to be bound totheory or mechanism, stimulation of erythropoiesis may be achieved bytwo pathways: a) by a direct effect on erythroid progenitors; and/or b)indirectly, by stimulating the function and production/secretion ofErythropoietin (EPO).

According to some embodiments, compounds represented by formula (I-a)are able to induce a dramatic and unexpected change in erythropoiesisand hemoglobin production. As detailed above, a co-drug represented byformula (I-a) may be hydrolyzed in-vivo to one or more active compounds,such as, for example, 5-ALA and a carboxylic acid compound (that may beactive as an HDACI). Moreover, as further exemplified hereinbelow, theeffect of the co-drug on inducing erythropoiesis and hemoglobinproduction in a target cell is enhanced as compared to introducing thecell with a 5-ALA or with an HDACI, or even compared to introducing thecell with a mixture of 5-ALA and HDACI. Thus, introducing a target cellwith a compound represented by formula (I-a) provide synergistic resultswith respect to erythropoiesis and hemoglobin production as compared tointroducing the cells with compounds such as, 5-ALA, HDACI or a mixturethereof.

According to some embodiments and as further exemplified herein below, aco-drug represented by formula (I-a) is able to induce a synergisticeffect on the synthesis of the protoporphirin IX (PpIX), which isgenerated in an early stage of the heme biosynthetic pathway. The effecton the synthesis of PpIX by a co-drug represented by formula (I-a) isenhanced as compared to the effect on the synthesis of PpIX exerted by5-ALA alone, HDACI alone or a mixture of 5-ALA and HDACI.

According to some embodiments and as further exemplified hereinbelow, aco-drug represented by formula (I-a) is able to induce the activity ofporphobilinogen deaminase (PBGD), which is a key rate-limiting enzyme inthe heme biosynthesis pathway. Evaluation of PBGD activity showed thatco-drug of 5-ALA and HDACI was more efficient in elevating PBGDactivity. The effect on the activity of PBGD by a co-drug represented byformula (I-a) is enhanced as compared to the effect on the activity ofPBGD exerted by 5-ALA alone, HDACI alone or a mixture of 5-ALA andHDACI.

According to some embodiments and as further exemplified hereinbelow, aco-drug represented by formula (I-a) is able to induce the expression ofFerrochelatase, which is the enzyme that catalyzes the final step in theheme biosynthetic pathway. Evaluation of Ferrochelatase proteinexpression showed that co-drug of 5-ALA and HDACI was more efficient inelevating Ferrochelatase activity. The effect on the activity ofFerrochelatase by a co-drug represented by formula (I-a) is enhanced ascompared to the effect on the activity of Ferrochelatase exerted by5-ALA alone, HDACI alone or a mixture of 5-ALA and HDACI.

According to further embodiments, and as further exemplifiedhereinbelow, a co-drug represented by formula (I-a) is able induce amarked increase of total heme content in treated cells. The increase inthe total heme content is significantly higher in cells introduced witha co-drug represented by formula (I-a), as compared to cells introducedwith 5-ALA alone, HDACI alone or a mixture of 5-ALA and HDACI.

According to additional embodiments, and as further exemplifiedhereinbelow, a co-drug represented by formula (I-a) is able to induceexpression of globin genes, such as, for example, the α-globin gene. Theelevation in expression of the globin genes is significantly higher incells introduced with a co-drug represented by formula (I-a), ascompared to the effect induced by 5-ALA alone, HDACI alone or a mixturethereof.

According to further embodiments, and as further exemplifiedhereinbelow, a co-drug represented by formula (I-a) is able to inducedifferentiation of erythroids, which may thereby result in increase inerythropoiesis due to increase in mature red blood cells.

According to some embodiments, there is thus provided a method for thetreatment or prevention of anemia, comprising the step of administeringto a subject in need thereof a compound represented by the structure offormula (I-a), or a pharmaceutical composition comprising such compound.

In some embodiments, there is provided a method for inducingerythropoiesis, comprising the step of administering to a subject inneed there of a compound represented by the structure of formula (I-a),or a pharmaceutical composition comprising such compound.

According to some embodiments, there is provided a method for thetreatment or prevention of anemia by inducing erythropoiesis, comprisingthe step of administering to a subject in need thereof a compoundrepresented by the structure of formula (I-a), or a pharmaceuticalcomposition comprising such compound.

According to some embodiments, the compounds of Formula (I) and Formula(I-a) may be prepared by various methods. For example, the compounds ofFormula (I) and Formula (I-a) may be produced according to the generalscheme presented in FIG. 1.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the compounds of the present invention, togetherwith suitable diluents, preservatives, solubilizers, emulsifiers,adjuvant and/or carriers. Such compositions are liquids or lyophilizedor otherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils).

Further included are particulate compositions coated with polymers(e.g., poloxamers or poloxamines). Other embodiments of the compositionsof the invention incorporate particulate forms, protective coatings,inhibitors or permeation enhancers for various routes of administration,including parenteral, topical, pulmonary, nasal and oral. In someembodiments, the pharmaceutical composition is administeredparenterally, paracancerally, transmucosally, transdermally,intramuscularly, intravenously, intradermally, subcutaneously,intraperitonealy, intraventricularly, intracranially or intratumorally.

Moreover, as used herein “pharmaceutically acceptable carriers” are wellknown to those skilled in the art and include, but are not limited tophosphate buffer and/or saline. Additionally, such pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions.

Parenteral vehicles may include, for example, sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent.

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system.

The pharmaceutical preparation may comprise one or more of the compoundsrepresented by the structure of formula (I) or formula (I-a), or mayfurther include a pharmaceutically acceptable carrier, and can be insolid or liquid form such as tablets, powders, capsules, pellets,solutions, suspensions, elixirs, emulsions, gels, creams, orsuppositories, including rectal and urethral suppositories.Pharmaceutically acceptable carriers include gums, starches, sugars,cellulosic materials, and mixtures thereof. The preparation can also beadministered by intravenous, intra-arterial, or intramuscular injectionof a liquid preparation, oral administration of a liquid or solidpreparation, or by topical application. Administration can also beaccomplished by use of a rectal suppository or a urethral suppository.

The pharmaceutical preparations can be prepared by known dissolving,mixing, granulating, or tablet-forming processes.

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as aerosols of the polypeptide delivered to the nasopharynxor as injectables, either as liquid solutions or suspensions, however,solid forms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.The active therapeutic ingredient is often mixed with excipients thatare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof.

In addition, if desired, the composition can contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, which enhance the effectiveness of the active ingredient.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule), which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, methanesulfonic, benzenesulfonic, naphthalene sulfonic, oxalic, tartaric, mandelic, and thelike. Salts formed from the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

In another embodiment, the active compound can be delivered in avesicle, such as, for example, a liposome.

For topical administration to body surfaces using, for example, creams,ointments, gels, lotions, solutions, co-solvent solutions, suspensions,and the like. The compounds of the present invention or theirphysiologically tolerated derivatives such as salts, hydrates, and thelike are conveniently prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

According to yet further embodiments, the compounds of formula (I) or(I-a) may be used in cosmetic treatments, wherein the compounds may beformulated for topical administration and may be administered to bodysurface of a subject. The body surface may include, for example, thesubject's skin. In some embodiments, the cosmetic treatment may includethe use of light irradiation that may be performed after the topicalformulation comprising the compounds of formula (I) or formula (I-a) areadministered to the body surface.

While a number of aspects and some embodiments have been discussedabove, those of skill in the art will readily recognize certainmodifications, permutations, additions and sub-combinations thereof. Itis therefore intended that the following appended claims and claimshereafter introduced be interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Example 1A Preparation of Compounds of Formula (I) and Formula(I-a)

The preparation of compounds of Formula (I) and formula (I-a) isperformed according to the scheme shown in FIG. 1:

Preparation of Chloroalkyl esters 4

To a stirred solution of an acyl chloride (compound 2 in FIG. 1, (R¹ isother than H)) and a catalytic amount of a Lewis acid (anhydrous zincchloride), under a nitrogen atmosphere, an aldehyde (compound 3 inFIG. 1) is dropwise added. The solution is stirred for several hourswhile the reaction progress is monitored by TLC/HPLC. Upon detection ofcomplete consumption of either the acyl halide (compound 2) or thealdehyde (compound 3), the reaction mixture is filtered through silicagel and the residual chloroalkyl ester product (compound 4) is purifiedby chromatography or distillation.

Preparation of Compound 6 (i.e., a compound of Formula (I) or (I-a)wherein R³=nitrogen protecting group):

To an equimolar mixture of a compound of Formula 5 (R³=nitrogenprotecting group such as Boc, CBZ etc.) and a chloroalkyl ester ofFormula 4, a tertiary base (triethylamine, N-methylmorpholine,ethyl-diisopropyl amine, 1,8-diazabicycloundec-7-ene (DBU), or4-dimethylaminopyridine) is added in a dry inert solvent (methylenechloride, methylethyl ketone, chloroform, toluene, ethyl acetate oracetonitrile). The mixture is stirred and heated while the reactionprogress is monitored by TLC/HPLC. Upon detection of completeconsumption of either compound of Formula 4 or compound of Formula 5,the reaction mixture is filtered, the filtered salt is washed with ethylacetate and the combined filtrate is evaporated. The residue isdissolved in ethyl acetate and is washed with saturated aqueous sodiumbicarbonate and brine, and the organic phase is dried over magnesiumsulfate or calcium chloride and the solvent is evaporated. The residueis purified by chromatography to give the product of Formula 6.

Preparation of Compound 7 (i.e., a compound of Formula (I) or (I-a)wherein R³=H):

An N-Boc-protected compound of Formula 6 is dissolved in ice-cold ethylacetate and is treated with a freshly prepared solution of an acid suchas hydrogen chloride in ethyl acetate. The reaction progress ismonitored by TLC/HPLC and upon detection of complete consumption of 6the solvent is evaporated to give the salt of formula (7).

When a CBZ-protected compound of Formula 6 is used, the CBZ group isremoved by Pd catalyzed hydrogenolysis in the presence of an acid HX(e.g., hydrochloric acid, HCl) dissolved in a suitable solvent(methanol). The reaction progress is monitored by TLC/HPLC and upondetection of complete consumption of 6 the mixture is filtered, and thefiltrate is evaporated to give the salt of formula (7).

Example 1B Preparation of exemplary compounds1-(Octanoyloxy)ethyl-5-Amino-4-oxopentanoate Hydrochloride (A);1-(Butyryloxy)butyl-5-amino-4-oxopentanoate Hydrochloride (B) and1-(Butyryloxy)propionyl-5-amino-4-oxopentanoate hydrochloride (C)

General Procedure A:

Coupling 5-(tert-Butoxycarbonylamino)-4-oxopentanoic Acid withChloroalkyl Esters. A mixture of5-(tert-butoxycarbonylamino)-4-oxopentanoic acid, (1.2 equiv.) and achloromethyl/chloroethyl ester (1 equiv.) in dry methyl ethyl ketoneunder N₂ was stirred while triethylamine or DBU (1.2 equiv) was addeddropwise. The mixture was refluxed (<80° C.) overnight when usingchloromethyl esters or for 2 days in the case of chloroethyl esters. Awhite precipitate which formed was filtered and washed with EtOAc, andthe filtrate was evaporated. The residue was dissolved in EtOAc and waswashed with saturated NaHCO₃ (×3) and brine (×3), dried over Na₂SO₄,filtered, and evaporated. The crude product was purified by flashchromatography.

General Procedure B:

Removal of N-tert-Boc Group. To an ice-cold solution of an N-Bocprotected compound in EtOAc was added a freshly prepared solution of 4 NHCl in EtOAc obtained by addition of acetyl chloride to a solution ofEtOH in EtOAc, or by gaseous HCl in dry ether. The ice bath was removedafter 1 h, and the solution was allowed to warm to room temperature. Thereaction was monitored by TLC (hexane/EtOAc, 2.5:1) and was generallycompleted within a few hours. The solvent was evaporated to give thecrude product. The latter was dissolved in MeOH, treated with activatedcharcoal, and filtered. The filtrate was evaporated and dried under highvacuum to give the product as a semisolid oil/foam.

Preparation of 1-(Octanoyloxy)ethyl-5-amino-4-oxopentanoateHydrochloride

The compound was prepared as described in Procedures A and B above from2-chloroethyloctanoate and 5-(tert-butoxycarbonylamino)-4-oxopentanoicacid. ¹H-NMR (300 MHz, CDCl₃) ppm δ 0.87 (t, J=7.20 Hz, 3H, CH2Me), 1.27(m, 8H, (CH₂)₄CH₂CH₂CO₂), 1.45 (d, J=5.40 Hz, 3H, OCH(CH₃)O), 1.58(sext, J=7.32 Hz, 2H, CH₂CH₂CH₂CO₂), 2.28 (t, J=7.30 Hz, 2H,CH₂CH₂CH₂CO₂), 2.66 (“t”, 2H, COCH₂CH₂CO₂), 2.94 (“t”, 2H, COCH₂CH₂CO₂),4.28 (s, 2H, CH₂NH₂), 6.83 (q, J=5.60 Hz, 1H, OCHO).

Preparation of 1-(Butyryloxy)butyl-5-amino-4-oxopentanoate Hydrochloride

The compound was prepared as described in Procedures A and B above from2-chlorobutylbutyrate and 5-(tert-butoxycarbonylamino)-4-oxopentanoicacid. ¹H-NMR (300 MHz, CD₃OD) ppm δ 0.95+0.96 (two t, J=6.6 Hz, 6H, twoMe), 1.65 (m, 6H, CH3CH₂CH₂CO₂+CH₃CH₂CH₂CH), 2.27 (t, J=10.8 Hz, 2H,CH₂CH₂CH₂CO₂), 2.70 (“t”, 2H, COCH₂CH₂CO₂), 2.87 (“t”, 2H, COCH₂CH₂CO₂),4.02 (s, 2H, CH₂NH₂), 6.72 (t, J=8.4 Hz, 1H, OCHO).

Preparation of 1-(Butyryloxy)propionyl-5-amino-4-oxopentanoateHydrochloride: The compound was prepared as described in Procedures A &B from 2-chloroethylbutyrate and5-(tert-butoxycarbonylamino)-4-oxopentanoic acid. ¹H-NMR (300 MHz,CD₃OD) ppm δ 0.95 (t, J=7.5 Hz, 6H, two Me), 1.61 (sextet, 2H, J=7.5 Hz,CH₃CH₂CH₂CO₂), 1.78 (quint, J=5.7 Hz, 2H, CH₃CH₂CH) (t, J=7.2 Hz, 2H,CH₃CH₂CH₂CO₂), 2.70 (m, 2H, COCH₂CH₂CO₂), 2.86 (m, 2H, COCH₂CH₂CO₂),4.04 (s, 2H, CH₂NH₂), 6.72 (t, J=5.7 Hz, 1H, OCHO)

Examples 2-14 Biological Effect of Compounds of Formula (I) and Formula(I-a) Materials and Methods Erythroid Cell Line

K-562, a human erythroid-like cell lines were derived from cellsexplanted from patients with chronic myelogenous leukemia at blastcrisis. When induced along the erythroid lineage, the cells accumulateHb, but fail to express the full erythroid phenotype. Such cells canserve as an experimental model for studying erythroid differentiationand hemoglobin (Hb) synthesis and accumulation at cellular and molecularlevels. The cells provide reproducible, uniform, large populations ofcells, which can undergo a synchronized differentiation program. Hence,the K-562 cell line may be used to reflect the effect of treatment onerythropoiesis.

Cell Cultures

Myelogenous leukaemia K562 and hepatoma Hep3B cells are grown on tissueculture plates (Greiner, Glos, UK) in RPMI-1640 medium supplemented with10% fetal calf serum, antibiotics (penicillin-streptomycin-nystatin),and L-glutamine (Biological Industries, Kibbutz Beit-Haemek, Israel).The cells were grown at 37° C. in a humidified atmosphere with 5% CO₂,and were re-cultured twice a week.

Human Glioblastoma U251 cells are grown on tissue culture plates(Greiner, Glos, UK) in RPMI-1640 medium supplemented with 10% fetal calfserum, antibiotics (penicillin-streptomycin-nystatin), and L-glutamine(Biological Industries, Kibbutz Beit-Haemek, Israel). The cells aregrown at 37° C. in a humidified atmosphere with 5% CO₂, and werere-cultured twice a week.

The murine mammary breast carcinoma 4T1 (CRL-2539) and embryonic ratheart H9C2 (CRL-1446) cell lines are obtained (ATCC, Rockville, Md.,USA). U251 MG human glioma cell line (Cyagen Bio9sciences) and normalhuman astrocytes (NHA, Lonza, International). Cells are grown in DMEMwith 10% FCS and 2 mM L-glutamine, except the astrocytes that are grownin ABM™ Basal Medium and AGM™ Bullet Kit® (Lonza, International). Allcells are grown in the presence of 100 units/mL penicillin, 100 μg/mLstreptomycin, 12.5 un/mL nystatin (Biological Industries Beit-Haemek,Israel), and incubated in a humidified atmosphere of 5% CO2 95% air at37° C.

Cytotoxicity:

Cytotoxicity is evaluated by dual fluorescent staining; propidium iodide(red fluorescence) as a marker for dead (or damaged) cells andfluorescein diacetate (green fluorescence) as a marker for metabolicallyactive cells.

Programmed Cell Death (Apoptosis):

Apoptosis is measured using specific antibodies conjugated to afluorescence probe (ApopTag, Oncor, Gaithersburg, Md.). Cells are scoredby flow cytometry.

Viability Assays:

MTT: Cells are grown in 24 wells plate in a volume of 0.5 mL and treatedas indicated for 96 hours. Then, 77 μL of 5 mg/mL MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, (Sigma,Israel)) is added to each well for 2 hours. Afterwards, 400 μL of DMFsolution (100 gr SDS dissolved in 250 mL dd H₂O with 250 mL of DMF) isadded for 4 hours. Absorbance is then measured in 570 nm using a Tecanspectrophotometer (NeoTec, Canada).

Hoechst viability assay is performed as described [12].

Intracellular Heme Assay:

The method for quantitative determination of total heme in cell lysatesis performed using hemin as a standard. The “total heme” or “endogenousheme” measured in these assays includes bound and free heme. Followingtreatments, 100 or 500 μg of cell lysate was added to 100 μLorthotolidine reagent (0.25 gr orthotolidine (Sigma-Aldrich, Israel)dissolved in 80 mL glacial acetic acid (Sigma-Aldrich, Israel) and 10 mLdd H₂O). Heme is oxidized to a green product by adding 100 μL of 1.2%H₂O₂ to the mixture for 10 minutes incubation in the dark. The firstoxidation product was further oxidized to a yellow product by adding 0.5mL of a 1:9 v/v solution of diluted acetic acid. Absorbance of thesecond product was read at 430 nm using a spectrophotometer (TecanTrading AG, Switzerland).

Assessment of Cellular PpIX:

Following treatment, cells are harvested, collected by centrifugation,washed twice and resuspended in 0.5 mL with sterile PBS. PpIXfluorescence is measured in 10,000 cells per sample using a FACS(Becton-Dickinson, CA, USA) with an excitation wavelength of 488 nm andemission wavelength >670 nm. Images of the stained cells are examinedusing a fluorescent microscope (IX70) (Olympus Tokyo, Japan) using anexcitation filter of 330-385 nm and barrier filter at 420 nm

Western Blotting:

Proteins are quantified and equalized using the Bradford assay (Bio-Rad,CA, USA) and resolved on a 12% polyacrylamide gel. Afterwards, proteinsare transferred from the gel onto nitrocellulose membranes (Bio-Rad, CA,USA) using a semi-dry transfer apparatus (Bio-Rad, CA, USA). Afterblocking the membranes with phosphate-buffered saline+0.2% Tween-20(PBST) (Sigma-Aldrich, Israel) and 5% skim-milk (BD-Diagnostic Systems,MD, USA), membranes are incubated with primary rabbit anti-human PBGDantibody (a generous gift from HemeBiotech, Sweden), or with rabbitanti-α-Hb (H80) (Santa Cruz Biotechnology, CA, USA), or withHRP-conjugated mouse anti-β-actin antibody (C4) (Santa CruzBiotechnology, CA, USA) diluted in blocking solution for 1 h in roomtemperature or for overnight in 4° c., washed with PBST and incubatedwith secondary antibodies diluted in blocking solution Immuno-reactiveproteins were visualized with enhanced chemiluminiscence detectionEZ-ECL kit (Biological Industries, Israel) used as recommended by themanufacturer.

Hb and Glycophorin a Immunostaining Analysis by Flow Cytometry:

K562 cells are fixed and stained with primary antibodies anti-α Hb oranti-Glycophorin A and are tagged using the compatible secondaryantibodies-Alexa 488-conjugated anti-rabbit antibody donkey anti-goat(Invitrogen, Oregon, USA) as recommended by Cell Signaling (MA, USA).Fluorescence is measured in 10,000 cells using FACS with an excitationwavelength of 488 nm and emission wavelength of 530 nm. K562 cells weresuspended in paraformaldehyde 4% for 10 min at 37° C., followed byincubation with chilled methanol (final concentration 90%) for 30 min onice. Cells were washed twice with PBS−/− and blocked with BSA 0.5% inPBS−/− for 10 min at room temperature. Afterwards, the cells wereincubated in 50 μL of the primary antibody goat polyclonalFerrochelatase (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) orgoat polyclonal glycophorin A (Santa Cruz Biotechnology, Santa Cruz,Calif., USA) (1:50 in BSA 0.5%) or anti-α Hb for 30 min at roomtemperature, washed once with PBS−/−, and incubated with a fluorescentdonkey anti-goat IgG secondary antibody (1:1000) (Invitrogen, Carlsbad,Calif., USA) for 30 min at room temperature. The cells were washed againwith PBS−/− and fluorescence was measured in 10,000 cells per sampleusing a FACS (Becton-Dickinson, San Jose, Calif., USA) with anexcitation wavelength of 488 nm and emission wavelength 530 nm.

RNA Preparation and Concentration Determination

Total RNA was isolated after the cells were harvested using EZ-RNA totalRNA isolation kit according to the manufacturer's instructions(Biological Industries). RNA concentration was measured using NanoDropspectrophotometer.

Globin RNA Accumulation:

Globin RNAs are quantified by quantitative real time polymerase chainreaction, (qRT-PCR) using SYBR I Green (Applied Biosystems). The cDNA isgenerated from cell culture or tissues using a PCR purification kit(Qiagen, Valencia, Calif., USA). The qRT-PCR is performed in triplicateand is repeated in at least three separate experiments.

Quantitative PCR Amplification

Total RNA (1 μg) was primed by oligo dT and reverse-transcribed by VersocDNA kit according to the manufacturer's protocol (Thermo Scientific,West Palm Beach, USA). Quantitative PCR was done on a Step One Plusthermocycler (Applied Byosystems, Van Allen Way, CA, USA). Thecomparative threshold method was used to calculate the relative geneexpression. Values were normalized against glyceraldehyde 3-phosphatedehydrogenase (GAPDH) for the different genes. Real time PCR wasperformed with Syber Green (Kapa Biosystems, Woburn, Mass., USA) usingthe following primers:

TABLE 1 HBA1/ Forward 5′-CCGACAAGACCAACGTCA (SEQ ID NO. 1) A2 Reverse5′-CGAAGTGCGGGAAGTAGG (SEQ ID NO. 2) PBGD Forward 5′-ACGAGCAGCAGGAGTTCA(SEQ ID NO. 3) Reverse 5′-ATGTCCTGGTCCTTGGCT (SEQ ID NO. 4) GAPDHForward 5′-CTTTGGTATCGTGGAAGGACTC (SEQ ID NO. 5) Reverse5′-AGTAGAGGCAGGGATGATGTTC (SEQ ID NO. 6)

Relative quantification of gene expression was determined by thecomparative threshold method (ÄCT), as described previously (Livak KJ,Schmittgen TD). Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(−Delta Delta C(T)) Method [18].Expression of the Gapdh mRNA in each individual sample was used tonormalize the dataset.

Hemoglobin Measurements

Samples are added to Drabkin's solution and the hemoglobin content isanalyzed colorimetrically at 540 nm in a spectrophotometer (Shimadzu UV1240). Hemoglobin content calculated curve following manufacturer'sinstructions.

Erythropoietin (EPO) Measurements

EPO protein level in hepatoma cell line and in kidney is determined byWestern blot analysis using rabbit polyclonal IgG antibody against EPO(Santa Cruz, USA) is resolved on a 12% polyacrylamide gel as describedabove. EPO RNA quantitation: RNA is extracted from cells and subjectedto qRT-PCR analysis using StepOnePlus (Applied Biosystem).

PBGD Enzymatic Activity Assay

K562 cells (1×10⁶) are treated for 96 hours. PBGD activity is assayed aspreviously described [15] by determining the fluorescence ofuroporphyrin formed by the light-induced oxidation of uroporphyrinogen,which is the immediate product of the enzymatic deamination of 4porphobilinogen molecules. In short, cells are harvested followingtreatment, and washed twice with PBS. After centrifugation, the pelletwas resuspended in 200 μL 50 mM Tris buffer (pH 8.2) with 0.2% Triton.The cells are homogenized on ice and protein levels are quantified usingthe Bradford method. 400 μg protein was taken out in duplicates forincubation in 300 μL of 50 mM Tris buffer (pH 8.2) containing 0.2%Triton, with final concentration of 85 μM PBG (Sigma, Israel) for 1 hourat 37° C. The reaction is stopped by addition of 1.2 mL 15.6% TCA. Thetubes are left open for room light exposure at room temperature for 10minutes in order to oxidize uroporphyrinogen to uroporphyrin. After 15minutes centrifugation at 3,300 rpm, 200 μL the supernatant wascollected into a black 96 well plate (Greiner Bio one, Germany). Thesamples fluorescence is read using a Synergy spectrofluorometer (BioTekInstruments, VT, USA) with an excitation wavelength of 404 nm, and anemission wavelength of 595 nm. PBGD specific activity is calculated asactivity percentage of control.

HDAC Activity

Inhibition of the activity HDAC class I and II in the cells is performedwith the fluorescent kit (AK-503, Biomol, USA). The cells are seeded(10×10³ cells/well) in 96-well plates (in quadruplicates) in growthmedium for 24 hours and then treated. Incubation with the HDAC substrate(Fluor de Lys™) is for 2 hours. The reaction is terminated by theaddition of Fluor de Lys™ developer and 2 μM trichostatin A (TSA). The %of inhibition is calculated from the ratio of the fluorescence (measuredat 355 nm excitation and 460 nm emission) in the treated compared to theuntreated control culture.

Histone Acetylation

Quantitative analyses of total Histone (H4 or H3) or specific histoneacetylation detection are conducted fresh from frozen tissues, andcultured adherent and suspension cells are conducted using Western blotanalysis for total H4 or H3 acetylation is performed as described [9]and for specific histone acetylation a fluorometric kit (Epigenetek,USA) is used.

Photosensitization of the Cells

U251 cells are seeded and incubated with tested compounds for 4 hours inserum-free medium. Cells are irradiated for 10 min using aVilber-Lourmat light source VL-206BL, delivering a power density of 13J/cm² at 360-410 nm (max at 365 nm).

Measurement of ROS

ROS are measured in live cells as intracellular peroxides by monitoringthe oxidation of DCF-DA. The membrane-permeable dye undergoesdeacetylation by intracellular esterases and oxidation by ROS. Cells(2×10⁵) are treated with the tested compounds for 4 hours, and then halfthe samples are irradiated for 10 min. The samples are incubated withDCF-DA (10 μM) for 30 min at 37° C., washed twice with PBS, and analyzed(10⁴ cells) by flow cytometry.

Assessment of Mitochondrial Membrane Potential (ΔΨm)

The fluorescent mitochondrial-specific cationic dye JC-1 undergoespotential-dependent accumulation in the mitochondria. U251 cells (2×10⁴)are seeded in 96-well black plates (Greiner Bio-One, Germany), treatedfor 4 hours with the tested compounds, and exposed to light irradiation(10 min) Immediately after irradiation, the mitochondrial membranepotential of the cells is measured as previously described [16]. Imagesof the stained cells were examined using a fluorescent microscope (NikonTE-2000E), an excitation filter of 450 nm, and a barrier filter at 520nm.

Apoptosis Assay by FACS Analysis

U251 or K562 cells (2×10⁵) are seeded in 60 mm plates and treated withthe tested compounds. After an incubation of 4 hours, half of thesamples are irradiated for 10 min, and 24 hours later, the cells aretrypsinized, double stained with Annexin V-FITC and PI (MEBCYTOApoptosis kit, MBL, Nagoya, Japan) according to the manufacturer'sinstructions, and subjected to flow cytometry analysis (FACSCaliburcytometer, Becton Dickinson, N.J.). The percentage of cells is definedby their distribution in a fluorescence dot plot using the flowcytometry analysis software-FlowJo.

Giemsa Staining

U251 cells are seeded on 6 well plates and treated with the testedcompounds. Four hours later, half of the samples are irradiated, and 1hour subsequently the cells were stained with May Grunwald for 5 min,washed with distilled water, and stained for 10 min with Giemsa stain(Sigma-Aldrich, st.louis, MO), prepared as per the manufacturer'sinstructions. After the cells are washed with distilled water, they areair-dried and observed under a light microscope.

Proteosome Activity Assay

U251 cells (10⁶) are seeded in 10 cm² cell culture dishes for 24 hoursand then treated with tested compounds for 4 hours and irradiated atlight intensity 6.5 J/cm². The cells are harvested with rubber policemenin PBS, centrifuged, and washed with PBS, and the pellets wereresuspended in 300 μL of Tris-base buffer (100 mM, pH 7.5) containing 1%Triton X-100 and homogenized on ice. The lysates (500 μg) are incubatedfor 1 hour with N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (13μM) (Sigma, St. Louis, Mo.) to measure the chemotrypsin-like activity ofthe proteasome and Z-Leu-Leu-Glu-β-naphtylamide (100 μM) (Sigma, St.Louis, Mo.) to measure the caspase-like activity of the proteasome. Thereaction is stopped by addition of ice-cold ethanol, and the samples arecentrifuged for 5 min at 5000 g. The supernatants are collected, and thefluorescence at excitation/emission filters of 390/460 nm was measuredby the FluoStar fluorimeter (BMG Labtech, Germany).

In Vivo Studies

In vivo normal Balb-c mice are induced by treatment with the hemolyticagent, phenylhydrazine (Sigma) dissolved at 6 mg/ml in PBS and injectedintraperitoneally at 60 mg/kg on two consecutive days. Pre-existing RBCsdecayed in the control mice within a week and started to recover by day4 and all survived and regained a normal hematocrit by day 10. Positivecontrol were mice treated with recombinant human EPO injectedintraperitoneally (50 IU/mouse) on 3 consecutive days, tested compoundswere injected to the mice at ½; ¼; ⅛; of their MTD. Blood samples drawnat different times points from mice by retro-orbital venipuncture underanesthesia. Hb level, number of red blood cell and reticulocytes, serumEPO and Hb content are determined.

In an additional setting 8 weeks old male Balb-c mice were divided totwo groups, Group 1 received 4 mg/kg ip dose of Doxorubicin (Dox) once aweek and Group 2 received the same Doxorubicin treatment and in additionthe mice were treated with 50 mg/kg ip dose of the tested compound threetimes a week. Termination was done on day 19, blood samples were drawnby retro-orbital venipuncture under anesthesia. Hemoglobin levels inheparinized whole blood were determined using Drabkin's reagent andstandard hemoglobin obtained from Sigma-Aldrich and prepared and storedas instructed by the manufacturer.

Syngeneic Murine 4T1 Breast Carcinoma Metastatic Model

Eight- to ten-week old female BALB/c mice are implanted sc with 4T1mammary carcinoma cells (5×10⁵). Tumor volume was measured with acaliper twice a week and calculated by measured lengths (L) and widths(W) using the (L×W²)/2 formula. Treatment commenced when the tumorvolume reached a 50-100 mm³ The mice are randomly assigned to treatmentgroups.

Flank Glioblastoma Xenograft Model

Eight- to ten-week old male HSD athymic FOX Nude mice (Harlan, Israel)are inoculated subcutaneously (sc) in the flank with 5×10⁶ U251 cells.Treatment commenced when the tumor volume reached a 50-100 mm³ The miceare randomly assigned to treatment groups.

Mice Treatment

The mice are randomized and divided to two arms, one arm is irradiated 3times/week together with the administration of the tested compounds andthe other arm is not. The tested compounds are given at 50 mg/kg and 25mg/kg by gavage 3 times/week. Tumor volume is measured with a calipertwice a week and calculated by measured lengths (L) and widths (W) usingthe (L×W²)/2 formula.

Photoirradiation Procedure

A high intensity light delivery system (Vario Ray, SeNET Haifa Israel)for PDT, is used as a light source. The wavelength range (600-700 nm),light energy density per pulse (0.6 J cm⁻²). Prior to thephotoirradiation, the mice are anesthetized and are then placed in aspecial plastic tube. The area of the sc implanted tumor is exposedthrough a hole and is irradiated. All animal experiments are conductedaccording to the NCI Laboratory Animal Care Guidelines and with theapproval of the Tel Aviv University Committee for Animal Experimentationand the Israel Ministry of Health.

Scanning Electron Microscopy and Transmission Electron Microscopy

Analysis is performed on bone-marrow, spleen and liver, processed asdescribed in [17]. Briefly, cells are treated for 96 hours (hrs),harvested and fixed with Karnovsky fixative. The samples are washed in0.1 M cacodylate buffer and fixed with 1% OsO₄ in 0.1 M sodiumcacodylate buffer for 1 hour (h). The samples are then dehydrated ingraded ethanol solutions and propylene oxide and embedded in agar mix.Thin sections are cut, stained with uranyl acetate and lead citrate, andobserved under a transmission electron microscope.

Immunohistochemistry (IHC) Staining

Immunohystochemistry (IHC) staining of paraffin-embedded blocks of bonemarrow, spleen and liver is performed as described [11] and stained forstem/progenitor cell marker CD133, cytochrome-c, Ki67, Epo receptor andc-Kit.

Example 2 Changes in Synthesis of PpIX in 1(562 Cells in Response toVarious Treatments

K562 cells are grown as described above and incubated without (control)or with 0.5 mM of various compounds for 96 hours with 5-ALA, HDACIcompound (in this example, butyric acid (BA)), a mixture of 5-ALA and anHDACI (BA); and a compound of Formula (I-a) (in this example,1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, a compound of formula (D)(hereinafter, AlaAcBu)). PpIX fluorescence is measured at the FL-3channel of FACS Calibur. Additionally, pictures of cells are taken usinga fluorescent microscope-Nikon TE200-E, to show PpIX fluorescence. Theresults are presented in FIGS. 2A-B. FIG. 2A shows a representativehistogram depicting the flow cytometry analysis of the various compoundstested. FIG. 2B shows the pictures of cells under various experimentalconditions. The results show that the effect exerted by a compound offormula (I-a) (in this example, AlaAcBu), on the intensity of thefluorescence generated by PpIX is substantially higher when compared tothe effect exerted by 5-ALA alone, HDACI alone (in this example, BA) orthe mixture of the 5-ALA and the HDACI (BA). The results thus suggestthat a co-drug of Formula (I-a) (in this example, AlaAcBu), which may behydrolyzed in the cells to 5-ALA and HDACI (in this example, BA),induces a synergistic effect on the synthesis of protoporphirin IX(PpIX), as compared to the effect by 5-ALA alone, HDACI (BA) alone oreven a mixture thereof.

Example 3 Activation of the Key Enzymes in the Heme Biosynthesis Pathway(PBGD and Ferrochelatase) in K562 Cells in Response to VariousTreatments

K562 cells are grown as described above and incubated with 0.5 mM ofvarious compounds for 96 hours. Activity of PBGD is evaluated asdescribed above. Expression level of the PBGD protein is evaluated usingWestern blot analysis (as detailed above). Expression level of PBGD mRNAare evaluated using Quantitative real-time PCR. The results arepresented in FIGS. 3A-C, respectively. As shown in FIG. 3A, which show abar graph of the relative activity of PGBD compared to control, theeffect of the compound of formula (I-a) (in this example,1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu)), on the activityof PGBD is significantly (**=p<0.05, *=p<0.1) higher compared to theeffect on the activity of PGBD exerted by ALA alone, HDACI alone (inthis example, BA), or a mixture of ALA and HDACI.

As shown in FIG. 3B, the expression levels of PGBD are increasedsimilarly after 96 hours with all three treatments (i.e. a compound offormula (I-a) (in this example,1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu)), ALA+HDACI (inthis example, BA) and HDACI (BA), compared to untreated cells or cellstreated with ALA. The results thus suggest that a co-drug of formula(I-a) (in this example, 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate,(AlaAcBu)), which may be hydrolyzed in the cells to 5-ALA and HDACI (BAin this example), induces a synergistic effect on the synthesis andactivity of PGBD, as compared to the effect by 5-ALA alone, HDACI alone(BA in this example) or a mixture thereof. As shown in FIG. 3C, whichdemonstrates quantitative real-time PCR analysis of PBGD mRNA, all theindicated treatments elevated PBGD to the maximum level already after 24hours incubation (FIG. 3 C). After 48-96 hours PBGD levels following ALAand ALA+HDACI (BA in this example) treatment significantly decreased.These results may indicate that the enhanced activity of PBGD has agreater effect on heme synthesis rather than on ifs mRNA or proteinexpression. As detailed above, since PBGD, which is the third enzyme inthe heme biosynthesis pathway, is a rate limiting enzyme in the hemebiosynthesis pathway, upregulation of the PBGD activity results inincreased biosynthesis of PpIX. The elevated levels of PpIX, result inincreased heme levels, which is necessary for hemoglobin synthesis.

Ferrochelatase is the enzyme that catalyzes the final step in the hemebiosynthetic pathway. Reference is now made to FIGS. 3D-E, which shows abar graph and FACS analysis of expression of Ferrochelatase proteinunder different experimental conditions, respectively. As shown in FIGS.3D-E, of formula (I-a) (in this example, AlaAcBu) elevatedFerrochelatase protein expression to a significantly higher level thandid ALA, HDACI (in this example, BA) or their mixture. The results thusindicate that AlaAcBu induces an efficient erythroid differentiationalso through activation of the key enzymes in the heme synthesispathway, namely, PBGD and Ferrochelatase.

Example 4 Synthesis of Hemoglobin in K562 Cells in Response to VariousTreatments

K562 cells are grown as described above and incubated with 0.5 mM ofvarious compounds for 96 hours. Total heme content in the cells isevaluated. In addition, a globin mRNA and protein expression levels areevaluated using Quantitative real-time PCR and Western blot analysis,respectively. The results are presented in FIGS. 4A-E. As shown in FIG.4A, which shows a bar graph of the fold increase in total heme content,the effect of the compound of formula (I-a) (in this example,1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu)) on the increaseof total heme is significantly (**=p<0.005) higher compared to theeffect on heme, exerted by ALA alone, HDACI alone (BA in this example)or a mixture of ALA and HDACI (BA). Reference is now made to FIG. 4B,which shows a bar graph of the fold increase in total heme content ascompared to control, in response to treatment with 0.25 nM (for 96hours) with various compounds of formula (I-a): (D), treatment with1-(butyryloxy)ethyl 5-amino-4-oxopentanoate (AlaAcBu); (C) treatmentwith 1-(butyryloxy)propionyl-5-amino-4-oxopentanoate hydrochloride, (B)treatment with 1-(butyryloxy)butyl-5-amino-4-oxopentanoatehydrochloride). The results show that the various compounds of formula(I-a) cause 15-22 fold increase of total heme as compared to the control(i.e. cells treated only with vehicle). As shown in FIG. 4C,Quantitative real-time PCR analysis of α-globin demonstrates that thecompound of formula (I-a) (in this example, AlaAcBu) significantlyelevates α-globin mRNA levels compared to untreated cells and cells thatwere treated with ALA, HDACI (in this example, BA) or the ALA+HDACImixture. α-Globin mRNA levels were examined following 24, 48 and 72hours incubation and reached their peak after 72 hours in which thecompound of formula (I-a) (in this example, AlaAcBu) elevated α-globinexpression 11.3-fold, significantly (p<0.5) more than did the ALA+HDACI(in this example, BA) mixture (9.3), HDACI (BA) (9.1) or ALA (2.2)alone. As shown in FIGS. 4D-E, which shows Western blot analysis of theprotein expression levels of α-globin and quantitation thereof,respectively, the protein expression levels of α-globin are markedlyelevated in response to incubating the cells with a compound of formula(I-a) (in this example, 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate,(AlaAcBu)) as compared to any other treatment. The results thus suggestthat a co-drug of formula (I-a), which may be hydrolyzed in the cells to5-ALA and HDACI, induces a synergistic effect on the total heme contentin the cells and on the expression of α-globin in the cells, as comparedto the effect by 5-ALA alone, HDACI alone or a mixture thereof.

Example 5 Differentiation of Erythroblasts in Response to VariousTreatments

K562 cells are grown as described above and incubated with 0.5 mM ofvarious compounds for 96 hours. Glycophorin A, which is asialoglycoprotein expressed on the surface of differentiatingerythroblast in the process of maturation to red blood cells is a markerof differentiation. Glycophorin A content is measured usingimmunostaining analyzed by flow cytometry, as detailed above. Theresults are presented in FIGS. 5A-B. As shown in FIG. 5A, which shows abar graph of the relative expression of Glycophorin A (as percentage ofcontrol), that a co-drug of formula (I-a) (in this example,1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu)) induces thehighest increase in the relative expression of the glycophorin A proteinas compared to any other treatment. The results represent average valuesobtained from >3 independent experiments. FIG. 5B shows a representativehistogram depicting the flow cytometry analysis. The results thussuggest that a co-drug of formula (I-a) (in this example, AlaAcBu),which may be hydrolyzed in the cells to 5-ALA and HDACI (in thisexample, BA), induces a synergistic effect on the hematopoieticdifferentiation of the cells (as determined by expression of GlycophorinA), as compared to the effect by 5-ALA alone, HDACI alone or a mixturethereof.

Example 6 Proliferation Arrest Induced by a Compound of Formula (I-a)

To evaluate the effect of various tested compounds on cellproliferation, K562 cells were treated for 96 hours and their viabilitywas evaluated by MTT as detailed above. The results are presented inFIG. 6A, which shows a bar graph illustrating the mitochondria activity(% of control) under various treatments. As shown in FIG. 6A, a compoundof Formula (I-a) (in this example,1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu)) as well as amixture of ALA+HDACI (in this example, BA), significantly inhibited cellproliferation (33% and 28% inhibition, respectively) while ALA did notaffect cell viability and HDACI (BA in this example) even increased it.The results thus indicate that the compound of Formula (I-a) (in thisexample, AlaAcBu) as well as a mixture of ALA+HDACI (in this example,BA), induce cell mortality, or alternately, decrease proliferation dueto cell differentiation. To distinguish between the two possibilities,the cells were double-stained with Annexin V and PI. The results areshown in FIG. 6B, which shows FACS analysis of the cells under differentexperimental conditions, as indicated. The mortality (necrosis, earlyand late apoptosis) assessed by FACS revealed that the amount ofnecrotic/apoptotic cells was unaffected by any of the treatments. Thus,the compounds of formula (I-a) (in this example, AlaAcBu) and theALA+HDACI (in this example, BA) mixture caused a cytostatic effectmanifested as proliferation arrest but not cell mortality.

Example 7 Cellular Maturation Induced by a Compound of Formula (I-a)

To evaluate morphologic changes associated with the effect of compoundof formula (I-a) (in this example, AlaAcBu) and the ALA+HDACI (in thisexample, BA) mixture on K562 cells, the cells were examined byimmunohystochemical experiments, as shown in FIG. 7. Typicalultrastructural changes induced by compound of formula (I-a) (in thisexample, AlaAcBu, panels E-F) and the ALA+HDACI (in this example, BA),panels C-D, identified by TEM included: chromatin condensation,cytosolic hemoglobinization (as shown for example, in FIG. 7, panelsC-D), formation of multiple vacuolar system preceding nuclear extrusion(as shown for example, in FIG. 6, panel E, (dashed arrow)) and centralstacking of mitochondria (as shown, for example, in FIG. 6 panels D, Eand F, solid arrows).

Example 8 An In-Vivo Effect of Compounds of Formula (I-a) on DoxorubicinInduced Anemia in Mice

In vivo 8 weeks old male Balb-c mice were divided to two groups, Group 1received 4 mg/kg ip dose of doxorubicin (Dox) once a week and Group 2received the same Dox treatment and in addition the mice were treatedwith 50 mg/kg ip dose of compound of formula (I-a) (in this example,1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu)) three times aweek. A scheme of the protocol of this experiment is shown in FIG. 8A.The experiment was terminated on day 19, blood samples were drawn byretro-orbital venipuncture under anesthesia. Hemoglobin levels inheparinized whole blood were determined using Drabkin's reagent andstandard hemoglobin obtained from Sigma-Aldrich and prepared and storedas instructed by the manufacturer. As shown in the bar graphsillustrated in FIG. 8B, the mice in Group 1 that were only treated withDox had a significantly lower level of hemoglobin in their blood ascompared to the Group 2 mice, which were administered with 50 mg/kg i.p.doses of a compound of formula (I-a) (in this example, (AlaAcBu)) twicea week, in addition to the Dox treatment. The results demonstrate thattreatment with a compound of Formula (I-a) can ameliorate theDox-induced suppression of erythropoiesis, in-vivo.

Example 9 Effect of Various Co-Drugs of Formula (I) and PDT onIntracellular Reactive Oxygen Species (ROS) Levels in Cancer Cells

U251 cells are incubated for 4 hours with 0.25 mM of any one of thefollowing: 5-ALA, an acyloxymethyl ester co-drug of formula (I); oracyloxyalkyl ester co-drugs of formula (I). Thereafter, half of thesamples are irradiated for 10 min (12.5 J/cm²). One hour afterirradiation, ROS is measured by the FL-1 channel of FACS Calibur, usingthe DCF-DA dye.

Example 10 Effect of Various Co-Drugs of Formula (I) and PDT onMitochondrial Membrane Potential and Mitochondrial Activity

U251 cells are treated with the indicated compounds for 4 hours with0.25 mM of any one of the following: 5-ALA, an acyloxymethyl esterco-drug of formula (I); or acyloxyalkyl ester co-drugs of formula (I),and irradiated. Mitochondrial activity is evaluated by MTT assay 24hours after light irradiation (12 J/cm²). The cells are furtherirradiated at differing light doses and the mitochondrial activity isevaluated by MTT assay 24 hours after light irradiation. Themitochondrial membrane potential of the cells is determined by stainingwith JC-1 immediately after irradiation (12 J/cm²). Photographs of thecells are taken with a Nikon TE200-E fluorescence microscope (Tokyo,Japan) using an excitation filter of 450 nm and a barrier filter at 520nm.

Example 11 Characterization of Cell Death Induced by Various Co-Drugs ofFormula (I)

U251 cells are exposed to 0.25 mM of any one of the following: 5-ALA, anacyloxymethyl ester co-drug of formula (I); or acyloxyalkyl esterco-drugs of formula (I). The samples are irradiated for 10 min after 4hours treatment. After 24 hours the cells are double-stained withAnnexin V-FITC and PI and analyzed by FACS. One hour after irradiation,the cells are stained with May Grunwald and Giemsa and observed underlight microscope-Nikon TE200-E.

Example 12 Cell Morphology of Cancer Cells Treated with Various Co-Drugsof Formula (I)

U251 cells are incubated for 4 hours with 0.25 mM of any one of thefollowing: 5-ALA, an acyloxymethyl ester co-drug of formula (I); oracyloxyalkyl ester co-drugs of formula (I), followed by lightirradiation for 10 min Cell morphology is examined after 24 hours by SEMand TEM electron microscopy.

Example 13 Proteasome Activity of Cancer Cells Treated with VariousCo-Drugs of Formula (I)

U251 cells are treated for 4 hours with 0.25 mM of any one of thefollowing: 5-ALA, an acyloxymethyl ester co-drug of formula (I); oracyloxyalkyl ester co-drugs of formula (I). Half of the samples areirradiated at light intensity 6.5 J/cm². One hour later, whole cellextracts (40 μg of protein/lane) are loaded and resolved on 12% SDS gel,and Western blot analysis is performed. The expression level of theproteins is tested with the appropriate mouse/rabbit antibody for eachprotein. The chemotrypsin-like activity of the proteasome is measured inthe treated cells and compared to that of untreated control cells. Thecaspase-like activity of the proteasome is measured in the treated cellsand compared to that of untreated control cells.

Example 14 Direct Effect of Octanoic Acid on Cellular HDAC Activity

In order to test the direct effect of octanoic acid, as well as co-drugsof formula (I-a) (which comprise octanoic acid as the carboxylic acid),on the activity of cellular HDAC, the activity of HDAC is evaluated byincubating U-251 cells for 2 hours with the cell-permeable HDACfluorometric substrate for HDAC class I and II (Fluor de Lys™), asdetailed above. The results, which are presented in FIG. 9A, show thatoctanoic acid inhibits HDACs of classes I and II with an IC50 of 500μM±20. As shown in FIG. 9B, incubating the cells with a co-drug offormula (I-a), inhibited the HDAC activity at IC50=35±3. The resultspresented in FIGS. 9A-B, demonstrate that octanoic acid is an HDACI.Additionally, the results presented in FIG. 9B demonstrate that thepotency of the compound of formula (I-a) comprising octanoic acid ishigher than the potency of the octanoic acid alone.

REFERENCES

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What is claimed is:
 1. A method for the treatment or prevention ofanemia, or for the induction of erythropoiesis comprising the step ofadministering to a subject in need thereof a composition comprisingHistone deacetylase (HDAC)-inhibitor and 5-aminolevulinic acid (5-ALA)or an ester thereof.
 2. The method of claim 1, wherein the ester is amethyl or a hexyl ester.
 3. The method of claim 1, wherein the HDACinhibitor is a carboxylic acid.
 4. The method of claim 3, wherein thecarboxylic acid is selected from the group consisting of pivalic,butyric, valeric, hexanoic, heptanoic, octanoic, decanoic,4-phenylbutyric, 4-phenylacetic and retinoic acid.
 5. The method ofclaim 3, wherein the carboxylic acid is butyric, octanoic, decanoic orvaleric acid.
 6. The method of claim 3, wherein the carboxylic acid isbutyric acid or octanoic acid.
 7. The method of claim 1, wherein thecomposition is in a form suitable for oral administration, parenteraladministration, topical administration, dermatological administration,administration by inhalation, or administration via a suppository. 8.The method of claim 1, wherein the composition is in a form suitable fororal administration, intravenous administration by injection, oradministration by inhalation.
 9. The method of claim 1, wherein the HDACinhibitor is an inhibitor of a Class I HDAC and/or a Class II HDAC. 10.The method of claim 1, wherein the HDAC inhibitor is a lysinedeacetylase inhibitor.